- Email: [email protected]
cardiodilatin (ANPCDD )—Immunoreactivity in the lacrimal gland of the domestic pig
Exp. EyeRes.(1990)
50, 313-316
Localization of Atrial (ANP/CDD)-lmmunoreactivity
Natriuretic Domestic
WINFRIED
LANGE”,
RUDOLF
E. LANG*,
Peptide/Cardiodilatin in the Lacrimal Gland Pig*
COELESTINA
BASTING
AND
JiiRGEN
of the W. UNGER”t
aDepartment of Anatomy, University of Munich, bDepartment of Pharmacology, University of Heidelberg, and cDepartment of Ophthalmology, University of Munich, F.R.G. (Received 14 June 1989 and accepted in revised form 13 October 1989)
The presence of atrial natriuretic peptic/cardiodilatin-immunoreactive material wasdemonstrated in the lacrimal gland of the domestic pig by high performance liquid chromatography and radioimmunoassay. The immunohistochemical localization revealed a distinct population of cuboid or spindle-shaped ANP/CDD-iR cells in the epitheiium of the terminal portion of the secretory tubules. In addition, a moderate number of positive ceiis was localized intraepitheiiaily in the intralobular ducts as well as the connective tissue between these ducts. Our findings provide a morphological indication that ANP/CDD may play a physiological role in the regulation of sodium transport and secretion in the lacrimal gland. Key words: lacrimal gland ; atriai natriuretic peptide/cardiodilatin : sodium secretion: immunocytochemistry : high performance liquid chromatography ; radioimmunoassay. 1. Introduction Atria1 natriuretic peptide/cardiodilati (ANP/CDD) is a peptide that has been demonstrated in special cardiocytes, predominently iu the right auricle of the mammalian heart (De Bold, 1979, 1985 ; De Bold et al., 1981; De Bold and Flynn, 1983; Forssmann et al., 1983, 1984; Forssmann, 1986). It causes a diuretic, natriuretic, and hypotensive effect, as well as an inhibition of renin and aldosteron secretion (De Bold et al., 1981; De Bold and Flynn, 1983; Atarashi et al., 1984; Burnett, Graujer and Opgennoth, 1984; Chartier et al., 1984; Cantin and Genes& 1985; Lang et al., 1985 ; Marin-Grez, Fleming and Steinhausen, 1986). Recent fmdings have indicated that ANP/CDD is present in endocrine cells of glands that are involved in’the regulation of water and sodium secretion, such as the salivary glands (Cantin et al., 1984 ; Metz, Mutt and Forssmann, 1984; Herbst et al., 1986) and the salt gland of the Peking duck (Lange et al., 1989). Since the lacrimal gland is secreting sodium into the lacrimal fluid in a concentration of 120-l 70 mm01 1-l (Reim, 1985) it became of interest to test the possibility that a peptidergic system may be present in lacrimal gland, comparable to other salt secreting glands that contain ANP/CDD. The goal of the present study was to demonstrate ANP/CDD in the lacrimal gland by high performance liquid chromatography (HPLC) and radioimmunoassay (RU), and ANP/CDD cells by immunocytochemistry (ICC). 2. Material Twenty
glands of domestic
pigs were
* Supported by the Walter-Schulz-Stifhmg. t For correspondence at: Department of Anatomy, University of Munich, Pettenkoferstrasse 11, D-8000 Miinchen 2, Federal Republic of Germany. 00144835/90/030313+04
Immunocytochemistry
By using the indirect peroxidase-antiperoxidase (PAP) technique (Sternberger, 19 79) ANP/CDDimmunoreactivity was demonstrated with the polyclonal antiserum API0 (Lang et al., 1985). Sections were incubated for 24 hr in antibody dilutions of 1: 200 to 1: 800 in Tris-Triton-Carrageenan buffer, pH 7.8 (Sofroniew, 1983). After several rinses in Tris buffer, the sections were incubated in a 1: 50 dilution of goat-antirabbit IgG for 30 min and, after additional rinses, for 1 hr in a 1: 75 dilution of PAP. The visualization of the antigen was carried out in a solution of 1 mg ml-’ diaminobenzidme in Tris buffer, containing O-01 y. hydrogen peroxide. Specificity tests were carried out either by omitting the ilrst antibody or be preincubation of the antibody dilution with a synthetic atria1 natriuretic factor l-28 (ANF l-28) at a concentration of 10 ,ug ml-‘. These procedures resulted in complete blockage of staining. HPLC and RIA
and Methods lacrimal
obtained from the local slaughter house. The organs were removed immediately after exsanguination and ten glands were hxed by immersion in Bouin’s fluid for immunocytochemistry. After fixation, the tissue blocks were dehydrated in alcohol, embedded in parafilnparaplast and 7-pm sections were cut on a sliding microtome. In addition, ten lacrimal glands were collected, frozen on dry ice and stored at - 70°C until processed for HPLC and RIA.
$03.00/0
The methods for extraction, chromatographic purification and radioimmunological measurements have been described previously in detail (Lang et al., 1985). In brief, reverse-phase HPLC was carried out on acidic extracts of porcine lacrimal glands by using 0 1990 Academic Press Limited
314
W. LANGE
ET AL.
FIG. 1. (a-g). Immunohistochemical staining of ANP/CDD in porcine lacrimal gland (Nomarski photomicrographs). (a) Crosssection through the terminal portions of the secretory tubules of the lacrimal gland. A small, densely stained, spindle-shaped cell is localized in the epithelium close to the basal lamina. (600 x ). (b) A larger, triangular positive cell in the connective tissue between the terminal portion of the secretory tubules (cross-section 1400 x ). (c) Single, small ANP/CDD-IR cells were observed in the epithelium of small intralobular ducts (cross-section, 400 x ). (d) Longitudinal section through a large intralobular duct, demonstrating a large immunoreactive cell in the basal part of the epithelium (800 x ). (e-g) Sections through intralobular ducts showing immunoreactive cells localized in the connective tissue close to the epithelium. (e, 500 x : f. 400 x , g, 5 50 x ).
ATRIOPEPTIOE
IN THE
PORCINE
LACRIMAL
GLAND
315
a linear gradient of water/acetonitrile from 20 to 50% over a period of 60 min, at a flow rate of 1 ml min-l. The chromatographic separation was carried out on a Bonda Pak Cl8 column (40 x 1 cm). The peptides were measured by UV-detection at a wavelength of 210 nm. Synthetic ANF 1-28 was used as standard. In addition, fractions of 1 ml were collected, lyophylized, reconstituted in assay buffer and measured for ANP/CDD-immunoreactivity by a specific and sensitive RIA (Lang et al., 1985).
3. Results lmmunocytochemistry
ANP/CDD-IR cells were localized in the epithelium as well as connective tissue of the pig lacrimal gland. Overall, the form of the immunoreactive cells was found to be variable, although the majority of cells was spindle-shaped or ovoid. Single located ANP/CDDIR cells were frequently found in the epithelium of the terminal portion of the tubular gland, closely localized to the basal lamina. These cells are characterized by a spindle-shaped morphological picture [Fig. 1(a)], although a few cells that are localized intraepithelially are smaller and show a cuboidal or ovoid shape. In no case were immunoreactive cells seen to contact the luminar site of the secretory tubules. In contrast to the distribution of these cells, a similar number of larger, mostly triangular ANP/CDD-IR cells were found in the connective tissue between the terminal portions of the secretory tubules [Fig. l(b)]. In addition, ANP/CDD-IR cells were observed in intralobular ducts of the lacrimal gland. Their distribution and number was similar to the cells in the terminal portion of the secretory tubules. Singlelocated ANP/CDD-IR cells were found in the epithelium of the intralobular ducts [Fig. l(c), (d)], as well as the connective tissue surrounding the ducts [Fig.
Ue-dl. HPLC and RlA
The chromatogram of pig lacrimal gland extracts obtained by reverse-phase HPLC with RIA detection is demonstrated in Fig. 2. The major peak was present near the elution site of ANF l-28, indicating that the immunoreactive material shows similar lipophilicity and molecular mass. When measured by RIA, the mean concentration of ANP/CDD-immunoreactive material was 813 + 150 pg g-l wet weight (mean + S.E.M.). Serial dilutions of extracts were parallel to the standard curve of the assay, 4. Discussion Our study demonstrates the presence of immunoreactive ANP/CDD in the lacrimal gland by HPLC and RIA. In addition, immunocytochemical staining 22
Fraction
FIG. 2. The chromatogram of an extract from porcine lacrimal gland, obtained by reverse-phase HPLC shows an immunoreactive peak near the elution site of synthetic ANF
l-28. The peptide concentration in the fractions was detected by RIA. revealed a system of ANP/CDD positive cells in several secretory and non-secretory parts of the gland. Although further characteristics of the immunoreactive cells, i.e. the definition of the exact cell type, remain to be clarified, our findings indicate that there may be an endocrine active system which may participate in the regulation of a constant outflow of sodium into the lacrimal fluid. In contrast to the physiologically varying sodium concentration of the salivary gland or the salt gland of the Peking duck, the tear fluid is characterized by a defined level of sodium. A constant sodium concentration is important for the physiological function of the lacrimal fluid to moisten the cornea and to avoid swelling of the cornea1 and conjunctival epithelium (Reim, 1985). The activity of the lacrimal gland is under regulatory influence of the autonomic nervous system (Stammer, 1964). In addition to dense cholinergic and catecholaminergic fibers (Huhatala et al., 19 7 7 ; Thorig et al., 1982), recent studies have demonstrated innervation of the lacrimal gland by peptidergic fibers, e.g. vasoactive intestinal polypeptide (VIP)- and substance P-immunoreactive fibers (Dartt et al., 1984; Niiinen et al., 1984). A recent study in the avian lacrimal gland has demonstrated colocalization between these peptides, as well as between acetylcholinesterase and VIP (Walcott, Sibony and Keyser, 1989). Functional studies have demonstrated that acetylcholine and VIP show a stimulatory effect on the secretory activity of the mammalian lacrimal gland (Dartt et al., 1984). In light of these studies, it is tempting to speculate that the distinct ANP/CDDcontaining cellular system within the lacrimal gland may contribute to the control of homeostasis during secretion of lacrimal fluid. In conclusion, our findings suggest that the sodium secretion in the lacrimal gland may be controlled by the interaction of the autonomic nervous system and the intrinsic ANP/CDD-IR system, which may serve as EER 50
W. LANGE
316
the endocrine part of the regulatory mechanisms. Although our studies do nut exclude the fact that additional factors may play a role in sodium transport and secretion in the lacrimal gland, our findings provide a morphological basis for a physiological function of ANP/CDD in the control of these secretory mechanisms. Acknowledgments The excellent technical and photographic assistance of Mrs I. Wild and MSS. Miihlsiemer is gratefully acknowledged. References Atarashi, K., Muh-ow, R. J., France-Saez, R., Snajdar, R. and Rapp, J. (1984). Inhibition of aldosterone production by au atrial extract. Science 224, 992-4. Burnett, J. C., Jr, Graujer, J. P., Dpgennoth, T. S. (1984). Effects of synthetic atrial natriuretic factor on renal function andrenin release.Am. J. Physiul. 247, F863-7. Cantin, M., Gutkowska, J., Thibault, G., Milne, R. W., Ledoux,S.,MinLi, S.,Chapeau,C., Garcia,R., Hamet,P. and Genest, J. (1984). Immunocytochemical localization of atria1natriuretic factor in the heart and salivary glands.Histochemistry80, 113-27. Cantin, M. and Genest,J. (1985). The heart and the atria1 natriuretic factor. En&r. Rev. 6, 107-27. Chartier, L., Schifli-in,E.,Thibault, G. and Garcia,R. (1984). Atria1 natriuretic factor inhibits the stimulation of aldosteronesecretion by angiotensin II, ACI’H and potassium in vitro and angiotensin II-induced steroidogenesis in vivo. Endocrinology115, 2026-8. Dartt, D. A., Baker,A. K., Vaillant, C. andRose,P. E.(1984). Vasoactiveintestinalpolypeptidestimulationof protein secretionfrom rat lacrimal gland acini. Am. J. Physiol. 247, G502-9. De Bold, A. J. (1979). Heart atria granularity: effects of changesin water-electrolyte balance. Pruc. Sot. Exp. Biul. Med. 161, 508-11. De Bold, A. J. (1985). Atria1 natriuretic factor: a hormone producedby the heart. Science 230, 767-70. De Bold, A. J., Boreinstein, H. B., Veress, A. T. and Sonnenberg,H. (1981). A rapid and potent natiuretic responseto intravenous injection of atrial myocardial extract in rats. Life Sci. 28, 89-94. De Bold, A. J. and Flynn, T. G. (1983). Cardionatrin I-A novel heart peptidewith potent diuretic and natriuretic properties.L$ Sci. 33, 297-302. Forssmann,W., Hock, D., Lottspeich, F., Henschen,A., Kreye, V.. Christmann, M., Reinecke, M., Metz, J., Carlquist,M. and Mutt, V. (1983). The right auricle of the heart is an endocrineorgan. Anat. Embryol. 168, 307-13. Forssmann,W. G., Birr, C., Carlquist, M., Christmann,M., Finke,R., Henschen,A., Hock, D., Kirchheim,H., Kreye,
ET AL.
V., Lottspeich,F., Metz, J., Mutt, V. and Reinecke,M. (1984). The auricular myocardiocytes of the heart constitute an endocrine organ. Characterization of porcine cardiac peptide hormone, cardiodilatin-126. Cell Tissue Res. 238, 425-30.
Forssmann,W. G. (1986). Cardiachormones.I. review on the morphology,biochemistryand molecularbiology of the endocrineheart. Eur. 1. Clin. Invest. 16, 439-51. Herbs& M., Goebel.J., Born, A. J. and Metz, J. (1986). Immunhistochemische Untersuchungen der Gangepitheliender menschlichenParotis.Abstract, 81. Versammlungder Anatomischen Gesellschaft,p. 71. Liibeck. Huhatala, A., Huikuri, K. T., Palkama. A. and Tervo, T. (1977). Innervation of the rat Harderian gland by adrenergicand cholinergicnerve fibers.Anat. Rec. 188, 263-72. Lang, R. E., Tholken, H., Ganten,D., Luft, F. C., Rushkoaho, H. and Unger,Th. (1985). Atria1 natriuretic factor - a circulating hormone stimulated by volume loading. Nature314, 264-6. Lange. W.. Unger, J., Weindl, A. and Lang, R. E. (1989). Demonstration of atria1 natriuretic peptide/ cardiodilatin (ANPICDD)- immunoreactivity in the salt gland of the Peking duck. Anat. Embryol. 179. 465-9. Marin-Grez, M., Fleming,J. T. and Steinhausen,M. (1986). Atria1 natiuretic peptide causes pre-glomerular vasodilatationand post-glomerularvasoconstrictionin rat kidney. Nature 324, 473-6. Metz, J., Mutt, V. and Forssmann, W. G. (1984). Immunohistochemicallocalization of cardiodilatin in myoendocrinecellsof the cardiac atria. Anat. Embryol. 170, 123-7. Nikkinen, A., Lehtosalo,J. I., Uusitalo,H., Palkama,A. and Panula, P. (1984). The lacrimal glandsof the rat and the guineapig areinnervatedby nerve fiberscontaining immunoreactivities for substanceP and vasoactive intestinalpolypeptide.Histochemistry 8 1, 2 3-7. Reim,M. (1985). In Augenheilkunde. P. 28. FerdinandEnke Verlag: Stuttgart. Sofroniew, M. V. (1983). Golgi-like immunoperoxidase stainingof neuronsproducingspecificsubstances or of neurons transporting exogenoustracer proteins. In Immunohistochemistry. Chapter 16. (Ed. Cuello, A. C.). Pp. 43147. IBRO,Raven Press:New York. Stammer, A. (1964). Ein Beitrag zur Struktur und mikroskopischenInnervation der HarderschenDriise der VGgel.Acta Univ. Szegedensis 10, 99. Sternberger, L. (1979). lmmunocytochemistry (2nd ed.) Wiley: New York. Thorig, L., Van Haeringen, N. J.. Timmermans,P. B. and Van Zwieten,P. A. (1982). Alpha-adrenoceptorcontrol of peroxidasesecretionfrom rat lacrimal gland cellsin vitro. Exp. Eye Res. 35, 2941. Walcott, B., Sibony, P. A. and Keyser, K. T. (1989). Neuropeptides and the innervation of the avian lacrimal gland. Invest. Ophthalmol. Vis. Sci. 30. 1666-74.
50, 313-316
Localization of Atrial (ANP/CDD)-lmmunoreactivity
Natriuretic Domestic
WINFRIED
LANGE”,
RUDOLF
E. LANG*,
Peptide/Cardiodilatin in the Lacrimal Gland Pig*
COELESTINA
BASTING
AND
JiiRGEN
of the W. UNGER”t
aDepartment of Anatomy, University of Munich, bDepartment of Pharmacology, University of Heidelberg, and cDepartment of Ophthalmology, University of Munich, F.R.G. (Received 14 June 1989 and accepted in revised form 13 October 1989)
The presence of atrial natriuretic peptic/cardiodilatin-immunoreactive material wasdemonstrated in the lacrimal gland of the domestic pig by high performance liquid chromatography and radioimmunoassay. The immunohistochemical localization revealed a distinct population of cuboid or spindle-shaped ANP/CDD-iR cells in the epitheiium of the terminal portion of the secretory tubules. In addition, a moderate number of positive ceiis was localized intraepitheiiaily in the intralobular ducts as well as the connective tissue between these ducts. Our findings provide a morphological indication that ANP/CDD may play a physiological role in the regulation of sodium transport and secretion in the lacrimal gland. Key words: lacrimal gland ; atriai natriuretic peptide/cardiodilatin : sodium secretion: immunocytochemistry : high performance liquid chromatography ; radioimmunoassay. 1. Introduction Atria1 natriuretic peptide/cardiodilati (ANP/CDD) is a peptide that has been demonstrated in special cardiocytes, predominently iu the right auricle of the mammalian heart (De Bold, 1979, 1985 ; De Bold et al., 1981; De Bold and Flynn, 1983; Forssmann et al., 1983, 1984; Forssmann, 1986). It causes a diuretic, natriuretic, and hypotensive effect, as well as an inhibition of renin and aldosteron secretion (De Bold et al., 1981; De Bold and Flynn, 1983; Atarashi et al., 1984; Burnett, Graujer and Opgennoth, 1984; Chartier et al., 1984; Cantin and Genes& 1985; Lang et al., 1985 ; Marin-Grez, Fleming and Steinhausen, 1986). Recent fmdings have indicated that ANP/CDD is present in endocrine cells of glands that are involved in’the regulation of water and sodium secretion, such as the salivary glands (Cantin et al., 1984 ; Metz, Mutt and Forssmann, 1984; Herbst et al., 1986) and the salt gland of the Peking duck (Lange et al., 1989). Since the lacrimal gland is secreting sodium into the lacrimal fluid in a concentration of 120-l 70 mm01 1-l (Reim, 1985) it became of interest to test the possibility that a peptidergic system may be present in lacrimal gland, comparable to other salt secreting glands that contain ANP/CDD. The goal of the present study was to demonstrate ANP/CDD in the lacrimal gland by high performance liquid chromatography (HPLC) and radioimmunoassay (RU), and ANP/CDD cells by immunocytochemistry (ICC). 2. Material Twenty
glands of domestic
pigs were
* Supported by the Walter-Schulz-Stifhmg. t For correspondence at: Department of Anatomy, University of Munich, Pettenkoferstrasse 11, D-8000 Miinchen 2, Federal Republic of Germany. 00144835/90/030313+04
Immunocytochemistry
By using the indirect peroxidase-antiperoxidase (PAP) technique (Sternberger, 19 79) ANP/CDDimmunoreactivity was demonstrated with the polyclonal antiserum API0 (Lang et al., 1985). Sections were incubated for 24 hr in antibody dilutions of 1: 200 to 1: 800 in Tris-Triton-Carrageenan buffer, pH 7.8 (Sofroniew, 1983). After several rinses in Tris buffer, the sections were incubated in a 1: 50 dilution of goat-antirabbit IgG for 30 min and, after additional rinses, for 1 hr in a 1: 75 dilution of PAP. The visualization of the antigen was carried out in a solution of 1 mg ml-’ diaminobenzidme in Tris buffer, containing O-01 y. hydrogen peroxide. Specificity tests were carried out either by omitting the ilrst antibody or be preincubation of the antibody dilution with a synthetic atria1 natriuretic factor l-28 (ANF l-28) at a concentration of 10 ,ug ml-‘. These procedures resulted in complete blockage of staining. HPLC and RIA
and Methods lacrimal
obtained from the local slaughter house. The organs were removed immediately after exsanguination and ten glands were hxed by immersion in Bouin’s fluid for immunocytochemistry. After fixation, the tissue blocks were dehydrated in alcohol, embedded in parafilnparaplast and 7-pm sections were cut on a sliding microtome. In addition, ten lacrimal glands were collected, frozen on dry ice and stored at - 70°C until processed for HPLC and RIA.
$03.00/0
The methods for extraction, chromatographic purification and radioimmunological measurements have been described previously in detail (Lang et al., 1985). In brief, reverse-phase HPLC was carried out on acidic extracts of porcine lacrimal glands by using 0 1990 Academic Press Limited
314
W. LANGE
ET AL.
FIG. 1. (a-g). Immunohistochemical staining of ANP/CDD in porcine lacrimal gland (Nomarski photomicrographs). (a) Crosssection through the terminal portions of the secretory tubules of the lacrimal gland. A small, densely stained, spindle-shaped cell is localized in the epithelium close to the basal lamina. (600 x ). (b) A larger, triangular positive cell in the connective tissue between the terminal portion of the secretory tubules (cross-section 1400 x ). (c) Single, small ANP/CDD-IR cells were observed in the epithelium of small intralobular ducts (cross-section, 400 x ). (d) Longitudinal section through a large intralobular duct, demonstrating a large immunoreactive cell in the basal part of the epithelium (800 x ). (e-g) Sections through intralobular ducts showing immunoreactive cells localized in the connective tissue close to the epithelium. (e, 500 x : f. 400 x , g, 5 50 x ).
ATRIOPEPTIOE
IN THE
PORCINE
LACRIMAL
GLAND
315
a linear gradient of water/acetonitrile from 20 to 50% over a period of 60 min, at a flow rate of 1 ml min-l. The chromatographic separation was carried out on a Bonda Pak Cl8 column (40 x 1 cm). The peptides were measured by UV-detection at a wavelength of 210 nm. Synthetic ANF 1-28 was used as standard. In addition, fractions of 1 ml were collected, lyophylized, reconstituted in assay buffer and measured for ANP/CDD-immunoreactivity by a specific and sensitive RIA (Lang et al., 1985).
3. Results lmmunocytochemistry
ANP/CDD-IR cells were localized in the epithelium as well as connective tissue of the pig lacrimal gland. Overall, the form of the immunoreactive cells was found to be variable, although the majority of cells was spindle-shaped or ovoid. Single located ANP/CDDIR cells were frequently found in the epithelium of the terminal portion of the tubular gland, closely localized to the basal lamina. These cells are characterized by a spindle-shaped morphological picture [Fig. 1(a)], although a few cells that are localized intraepithelially are smaller and show a cuboidal or ovoid shape. In no case were immunoreactive cells seen to contact the luminar site of the secretory tubules. In contrast to the distribution of these cells, a similar number of larger, mostly triangular ANP/CDD-IR cells were found in the connective tissue between the terminal portions of the secretory tubules [Fig. l(b)]. In addition, ANP/CDD-IR cells were observed in intralobular ducts of the lacrimal gland. Their distribution and number was similar to the cells in the terminal portion of the secretory tubules. Singlelocated ANP/CDD-IR cells were found in the epithelium of the intralobular ducts [Fig. l(c), (d)], as well as the connective tissue surrounding the ducts [Fig.
Ue-dl. HPLC and RlA
The chromatogram of pig lacrimal gland extracts obtained by reverse-phase HPLC with RIA detection is demonstrated in Fig. 2. The major peak was present near the elution site of ANF l-28, indicating that the immunoreactive material shows similar lipophilicity and molecular mass. When measured by RIA, the mean concentration of ANP/CDD-immunoreactive material was 813 + 150 pg g-l wet weight (mean + S.E.M.). Serial dilutions of extracts were parallel to the standard curve of the assay, 4. Discussion Our study demonstrates the presence of immunoreactive ANP/CDD in the lacrimal gland by HPLC and RIA. In addition, immunocytochemical staining 22
Fraction
FIG. 2. The chromatogram of an extract from porcine lacrimal gland, obtained by reverse-phase HPLC shows an immunoreactive peak near the elution site of synthetic ANF
l-28. The peptide concentration in the fractions was detected by RIA. revealed a system of ANP/CDD positive cells in several secretory and non-secretory parts of the gland. Although further characteristics of the immunoreactive cells, i.e. the definition of the exact cell type, remain to be clarified, our findings indicate that there may be an endocrine active system which may participate in the regulation of a constant outflow of sodium into the lacrimal fluid. In contrast to the physiologically varying sodium concentration of the salivary gland or the salt gland of the Peking duck, the tear fluid is characterized by a defined level of sodium. A constant sodium concentration is important for the physiological function of the lacrimal fluid to moisten the cornea and to avoid swelling of the cornea1 and conjunctival epithelium (Reim, 1985). The activity of the lacrimal gland is under regulatory influence of the autonomic nervous system (Stammer, 1964). In addition to dense cholinergic and catecholaminergic fibers (Huhatala et al., 19 7 7 ; Thorig et al., 1982), recent studies have demonstrated innervation of the lacrimal gland by peptidergic fibers, e.g. vasoactive intestinal polypeptide (VIP)- and substance P-immunoreactive fibers (Dartt et al., 1984; Niiinen et al., 1984). A recent study in the avian lacrimal gland has demonstrated colocalization between these peptides, as well as between acetylcholinesterase and VIP (Walcott, Sibony and Keyser, 1989). Functional studies have demonstrated that acetylcholine and VIP show a stimulatory effect on the secretory activity of the mammalian lacrimal gland (Dartt et al., 1984). In light of these studies, it is tempting to speculate that the distinct ANP/CDDcontaining cellular system within the lacrimal gland may contribute to the control of homeostasis during secretion of lacrimal fluid. In conclusion, our findings suggest that the sodium secretion in the lacrimal gland may be controlled by the interaction of the autonomic nervous system and the intrinsic ANP/CDD-IR system, which may serve as EER 50
W. LANGE
316
the endocrine part of the regulatory mechanisms. Although our studies do nut exclude the fact that additional factors may play a role in sodium transport and secretion in the lacrimal gland, our findings provide a morphological basis for a physiological function of ANP/CDD in the control of these secretory mechanisms. Acknowledgments The excellent technical and photographic assistance of Mrs I. Wild and MSS. Miihlsiemer is gratefully acknowledged. References Atarashi, K., Muh-ow, R. J., France-Saez, R., Snajdar, R. and Rapp, J. (1984). Inhibition of aldosterone production by au atrial extract. Science 224, 992-4. Burnett, J. C., Jr, Graujer, J. P., Dpgennoth, T. S. (1984). Effects of synthetic atrial natriuretic factor on renal function andrenin release.Am. J. Physiul. 247, F863-7. Cantin, M., Gutkowska, J., Thibault, G., Milne, R. W., Ledoux,S.,MinLi, S.,Chapeau,C., Garcia,R., Hamet,P. and Genest, J. (1984). Immunocytochemical localization of atria1natriuretic factor in the heart and salivary glands.Histochemistry80, 113-27. Cantin, M. and Genest,J. (1985). The heart and the atria1 natriuretic factor. En&r. Rev. 6, 107-27. Chartier, L., Schifli-in,E.,Thibault, G. and Garcia,R. (1984). Atria1 natriuretic factor inhibits the stimulation of aldosteronesecretion by angiotensin II, ACI’H and potassium in vitro and angiotensin II-induced steroidogenesis in vivo. Endocrinology115, 2026-8. Dartt, D. A., Baker,A. K., Vaillant, C. andRose,P. E.(1984). Vasoactiveintestinalpolypeptidestimulationof protein secretionfrom rat lacrimal gland acini. Am. J. Physiol. 247, G502-9. De Bold, A. J. (1979). Heart atria granularity: effects of changesin water-electrolyte balance. Pruc. Sot. Exp. Biul. Med. 161, 508-11. De Bold, A. J. (1985). Atria1 natriuretic factor: a hormone producedby the heart. Science 230, 767-70. De Bold, A. J., Boreinstein, H. B., Veress, A. T. and Sonnenberg,H. (1981). A rapid and potent natiuretic responseto intravenous injection of atrial myocardial extract in rats. Life Sci. 28, 89-94. De Bold, A. J. and Flynn, T. G. (1983). Cardionatrin I-A novel heart peptidewith potent diuretic and natriuretic properties.L$ Sci. 33, 297-302. Forssmann,W., Hock, D., Lottspeich, F., Henschen,A., Kreye, V.. Christmann, M., Reinecke, M., Metz, J., Carlquist,M. and Mutt, V. (1983). The right auricle of the heart is an endocrineorgan. Anat. Embryol. 168, 307-13. Forssmann,W. G., Birr, C., Carlquist, M., Christmann,M., Finke,R., Henschen,A., Hock, D., Kirchheim,H., Kreye,
ET AL.
V., Lottspeich,F., Metz, J., Mutt, V. and Reinecke,M. (1984). The auricular myocardiocytes of the heart constitute an endocrine organ. Characterization of porcine cardiac peptide hormone, cardiodilatin-126. Cell Tissue Res. 238, 425-30.
Forssmann,W. G. (1986). Cardiachormones.I. review on the morphology,biochemistryand molecularbiology of the endocrineheart. Eur. 1. Clin. Invest. 16, 439-51. Herbs& M., Goebel.J., Born, A. J. and Metz, J. (1986). Immunhistochemische Untersuchungen der Gangepitheliender menschlichenParotis.Abstract, 81. Versammlungder Anatomischen Gesellschaft,p. 71. Liibeck. Huhatala, A., Huikuri, K. T., Palkama. A. and Tervo, T. (1977). Innervation of the rat Harderian gland by adrenergicand cholinergicnerve fibers.Anat. Rec. 188, 263-72. Lang, R. E., Tholken, H., Ganten,D., Luft, F. C., Rushkoaho, H. and Unger,Th. (1985). Atria1 natriuretic factor - a circulating hormone stimulated by volume loading. Nature314, 264-6. Lange. W.. Unger, J., Weindl, A. and Lang, R. E. (1989). Demonstration of atria1 natriuretic peptide/ cardiodilatin (ANPICDD)- immunoreactivity in the salt gland of the Peking duck. Anat. Embryol. 179. 465-9. Marin-Grez, M., Fleming,J. T. and Steinhausen,M. (1986). Atria1 natiuretic peptide causes pre-glomerular vasodilatationand post-glomerularvasoconstrictionin rat kidney. Nature 324, 473-6. Metz, J., Mutt, V. and Forssmann, W. G. (1984). Immunohistochemicallocalization of cardiodilatin in myoendocrinecellsof the cardiac atria. Anat. Embryol. 170, 123-7. Nikkinen, A., Lehtosalo,J. I., Uusitalo,H., Palkama,A. and Panula, P. (1984). The lacrimal glandsof the rat and the guineapig areinnervatedby nerve fiberscontaining immunoreactivities for substanceP and vasoactive intestinalpolypeptide.Histochemistry 8 1, 2 3-7. Reim,M. (1985). In Augenheilkunde. P. 28. FerdinandEnke Verlag: Stuttgart. Sofroniew, M. V. (1983). Golgi-like immunoperoxidase stainingof neuronsproducingspecificsubstances or of neurons transporting exogenoustracer proteins. In Immunohistochemistry. Chapter 16. (Ed. Cuello, A. C.). Pp. 43147. IBRO,Raven Press:New York. Stammer, A. (1964). Ein Beitrag zur Struktur und mikroskopischenInnervation der HarderschenDriise der VGgel.Acta Univ. Szegedensis 10, 99. Sternberger, L. (1979). lmmunocytochemistry (2nd ed.) Wiley: New York. Thorig, L., Van Haeringen, N. J.. Timmermans,P. B. and Van Zwieten,P. A. (1982). Alpha-adrenoceptorcontrol of peroxidasesecretionfrom rat lacrimal gland cellsin vitro. Exp. Eye Res. 35, 2941. Walcott, B., Sibony, P. A. and Keyser, K. T. (1989). Neuropeptides and the innervation of the avian lacrimal gland. Invest. Ophthalmol. Vis. Sci. 30. 1666-74.