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Immunohistochemical identification and effects of atrial natriuretic peptide, pancreastatin, leucine-enkephalin, and galanin in the porcine pancreas
Peptides, Vol. 17, No. 3, pp. 503-509, 1996 Copyright 0 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/96 $15.00 + .OO
PIISO196-9781(96)00011-3
ELSEVIER
Immunohistochemical Identification and Effects of Atria1 Natriuretic Peptide, Pancreastatin, Leucine-Enkephalin, and Galanin in the Porcine Pancreas ERNEST ADEGHATE, * ’ ZSOLT EMBER,-/- TIBOR DONATH,? AND JAIPAUL SINGH$
DAVID J. PALLOT”
*Department of Human Anatomy, Faculty of Medicine and Health Sciences, United Arab Emirates University, P. 0. Box 17466, Al-Ain, United Arab Emirates fist Department of Anatomy, Semmelweis University Medical School, Tfizolto’ u. 58, Budapest, Hungary $Department of Applied Biology, University of Central Lancashire, Preston, England Received 5 November 1995 ADEGHATE, E., 2s. EMBER, T. DONATH, D. J. PALLOT AND J. SINGH. Zmmunohisfochemical ident$cafion and effecfs of atria1 nut&retie peptide, pancreastatin, leucine-enkephalin, and galanin in the porcine pancreas. PEPTIDES 17(3) 503-509, 1996.-This study demonstrates the presence and distribution of atrial natriuretic peptide (ANP) pancreastatin (PST), leucineenkephalin (Leu-IINK), galanin (GAL), and insulin in the pig pancreas. The effects of PST, ANP, Leu-ENK, and GAL on protein and amylase secretion were also investigated to determine their functional role in the control of pancreatic secretion. PST-immunoreactive cells were observed in the islet of Langerhans and in the wall of the ducts. Leu-ENK-immunopositive cells were observed in both the endo- and exocrine pancreas. It is colocalized with insulin in the islet of Langerhans. ANP immunoreactivity was discernible in nerve fibers and cells of the exocrine pancreas. GAL-immunopositive cells were observed in close association with insulin-posiblve cells in the islets of Langerhans and in the exocrine pancreas. Stimulation of isolated pancreatic segments with either ANP or Leu-ENK resulted in increased protein secretion and amylase output. The Leu-ENK-evoked amylase secretion was antagonized by naloxone. Pancreastatin was effective at all concentrations, but low concentration had more marked secretory effects whereas GAL failed to evoke any significant increases in either protein or amylase secretion. The results of the study have demonstrated a close association of peptidergic fibers with the secretory cells of the pancreas. The nerve fibers can release peptides that in turn can stimulate protein and amylase secretion. Porcine pancreas Atria1 natriuretic
Immunohistochemistry peptide
Leucine-enkephalin
THE innervation of the pancreas is provided mainly by the parasympathetic nerves ( 14,I 9,26) and to a lesser extent by the sympathetic nerves (14,24) of the autonomic nervous system. The main sources of innervat.lon include the vagus and the splanchnic nerves ( 14). The sites of nerve termination vary from species to species but in general they seem to terminate near blood vessels, basal membrane of pancreatic acinar and ductal cells, and islet cells ( 3,11,14). Stimulation of the parasympathetic nerves results in the release of acetylcholine (ACh), which in turn activates cholinergic muscarinic receptors. This in turn leads to the metabolism of membrane-bound phosphatidylinositol 1,4 bisphosphate (PIP*), resulting in the elevation of diacylglycerol (DG), inositol t&phosphate (II?,), and inositol tetrakisphosphate (IP,) to elicit secretion (4,5,8). DG stimulates protein kinase C
’ Requests
for reprints
should be addressed
to Ernest Adeghate,
Insulin
Galanin
Pancreastatin
whereas IP3 and IP, mobilize cellular calcium (Ca”). On the other hand, stimulation of adrenergic nerves results in the release of noradrenaline, which activates mainly p-receptors, resulting in the metabolism of adenosine 3,5 cyclic monophosphate (cyclic AMP), which mediates secretion (24). In addition to parasympathetic and sympathetic innervation of the pancreas, there is much evidence for the presence of noncholinergic, nonadrenergic, or peptidergic nerves that contain vasoactive intestinal polypeptide (VIP) in several animal species including rat, cat, dog, pig, and man (14,23,26). VIP elicits pancreatic secretion via cyclic AMP metabolism (23). Apart from VIP, immunohistochemical studies have demonstrated the presence of numerous neuropeptides, including gas&in-releasing peptide (GRP) , substance P, cholecystokinin, neuropeptide Y, calcitonin gene-re-
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ADEGHATE ET AL
IMMUNOHISTOCHEMICAL
IDENTIFICATION
lated peptide (CGRP) , and enkephalin-related peptides, mainly in the pancreas of rat and guinea pig (2,14,19,28,30). This study was designed to identify the distribution of atrial natriuretic peptide (ANP), pancreastatin (PST), leucine-enkephalin (LeuENK) ,and galanin (GAL) in the pig pancreas, especially because the gland has a similar innervation pattern to man. In addition, the study also investigated the effects of exogenous applications of ANP, PST, Leu-ENK, and GAL on total protein output and amylase secretion in the isolated pig pancreas to ascertain their functional roles in the stimulus-secretion coupling process. The effect of the gut peptide, cholecystokinin octapeptide (CCK-8), on amylase secretion was also investigated for comparison. METHOD
Immunohistochemistry All experiments were performed on segments of freshly slaughtered pig pancreas. Tissues were collected immediately after killing the animals and fixed for 48 h in a mixture of 2% formaldehyde and 15% of saturated aqueous picric acid solution in 0.1 M phosphate-buffered saline (PBS ) . The specimens were later immersed overnight in 20% sucrose solution and were subsequently sectioned into 40-50-km-thick slices using a vibroslice equipment (Chapden Instruments, USA). The sections were subsequently incubated for 48 h at 4°C with monoclonal guinea pig anti-insulin serum. After several washings in PBS (pH 7.4), the sections were incubated for 1 h at room temperature in biotinylated anti-rabbit immunoglobulin and later in avidin-biotinperoxidase complex. Sites of immunoreactions were detected using diaminobenzidine (DAB) solution (44 mg 300 ml -’ ) of PBS containing 0.04 ml of H202 (0.03%) for 3-5 min. A brown precipitate was observed in the sites of the antigen. After the DAB reaction, which indicates insulin-positive sites, the specimens were washed in PBS and incubated either in rabbit anti-GAL, anti-Leu-ENK, anti-ANP, or anti -PST. Incubation and washing periods were the same as for the insulin immunohistochemistry given above. Sites of immunoreaction were detected using diaminobenzidine solution (44 mg 300 ml-‘) of PBS containing 0.4% nickel ammonium sulphate and 0.04 ml of H,Oz (0.03%) for 3-5 min. The sites of unmunoreaction gave a dark-blue color. In some experiments, DAB only was used to identify PST and Leu-ENK. In all rinses PBS containing 0.5% Triton X-100 was used. After staining, the sections were counterstained with haematoxylin, dried, dehydrated, and mounted in De-PeX. (Serva, Heidelberg, Germany ) Sources of the Antisera ANP, PST, Leu-ENK, and GAL antibodies and antigens were bought from Peninsula Laboratories (California, USA). Inert and link antisera and the detecting reagents (avidin-biotin-peroxidase complex, DAB) were all products of DAK0 (Copenhagen, Denmark).
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Immunohistochemical
Controls
Control experiments included: i) primary antisera preabsorbed with 10e6 M of the corresponding peptide; ii) omission of the primary antisera followed by avidin-biotin-peroxidase complex staining; iii) substitution of the primary antisera with normal swine serum. These control incubations were carried out on sections adjacent to those used in the normal avidin-biotin-peroxidase complex procedure. In all these control experiments, immunolabeling was not observed. Specificity of the Antisera PST antibody has been shown by Lamberts et al. ( 18) and by Schmidt et al. (27) to be specific for PST, with no significant cross-reactivity with other peptides. GAL and Leu-ENK antibodies have been demonstrated by Adeghate and Singh ( 1) and by Schultzberg et al. (30) to be specific for GAL and Leu-ENK, respectively, with Leu-ENK having a 29% cross-reactivity with dynorphin A. ANP antiserum has also been shown to be specific for ANP and to not cross-react with any peptide (Peninsula Laboratories, 1993-94 catalogue). Measurements
of Protein Output
Immediately after killing the animals, the pancreas was removed and placed in an ice-cold Krebs Hesenleit solution (KH) and transported to the laboratory. The composition of KH in (mM) is: NaCl, 118; KCl, 4.76; CaCl*, 2.56; NaHC03, 25; KH2P04, 2.2; MgClz, 1.2, and glucose 10. The pancreas was cut into small segments (3-5 mg) and washed three times with 50 ml oxygenated KH solution (pH 7.4) maintained at 37°C to discard excess protein due to cutting the tissue. Tissue aliquots (approximately 30-40 mg) were placed in 5 ml plastic tubes containing control oxygenated KH solution (volume = 3 ml) and test KH solution (volume = 3 ml) containing different concentrations ( 10-13, 10-l’ M) of either ANP, GAL, Leu-ENK, or PST. The test tubes were incubated at 37°C in a shaking water bath for 20 min. After the incubation period the tissues were removed immediately from each test tube, blotted, and weighed. The concentration of protein in the supematant was determined by spectrotluorimetry using the Bradford (7) method. Bovine serum abumin (Sigma, UK) was used as standard. Protein output was expressed as mg ml-’ ( 100 mg tissue)-‘. Measurement
of Amylase Secretion
Portions (30-40 mg) of the pig pancreas were placed into 3ml aliquots of oxygenated KH solution containing different concentrations of either CCK-8 ( 10-‘2-10-‘o M), PST (lo-‘*lo-” M), ANP ( 10~“-10-9 M), GAL ( 10-“-10-9 M), or Leu-ENK ( 10-“-10-9 M). In some experiments the tissues were pretreated with 1O-4 naloxone for 5 min followed by incubation with Leu-ENK ( 10-“-10-9 M). In control experiments, pancreatic segments were incubated with either KH solution alone or with 10m4 naloxone. Samples were incubated at 37°C and constantly agitated for 20 min. At the end of the in-
FIG. 1. Light micrographs of haematoxylin-counterstained sections of the porcine pancreas. (a) Atria1 natriuretic peptide-immunoreactive neuron (arrow head), and fibers (arrow) in the exocrine pancreas and insulin-positive cells (brown reaction product) in the islet of Langerhans (i). x210. (b) Galanin (GAL)-immunopositive neuron (arrow) in the exocrine pancreas. Some GAL-positive cells (arrow head) lie within the islet (i). Insulincontaining cells are stained brown in the islet. x312. (c) Leucine-enkephalin (Leu-ENK)-immunoreactive cells (arrows) are distributed all over the exocrine pancreas. Leu-ENK is colocalized with insulin in some endocrine cells (open arrow). Insulin-containing cells are stained brown in the islet of Langerhans (i). X3 12. (d) Leu-ENK-immunoreactive neuron (arrow) in the interacinar areas. a, acinar tissue. X312. (e) Pancreastatin (PST)immunopositive cell (arrow) in the wall of a pancreatic duct (pt). X312. (f) PST-positive cells (arrow) in the islet of Langerhans (i). ~312. (a-c) Immunoreactivity developed with diaminobenzidine (DAB) and DAB/nickel ammonium sulphate. (d-f) Immunoreactivity developed with DAB alone.
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cubation amylase amylase amylase
period the supematant was decanted off and assayed for content by an on-line fluorimetric method (26) with LXas a standard. The tissues were blotted and weighed and expressed as units (U) ml-’ (100 mg tissue)-‘.
ET AL.
SEM protein output from pancreatic segments incubated with eitherANP(10-‘3-10-” M) [Fig. Zfb-d)],PSTf 10-‘3-10~” M) [Fig. 2(e-g)], Leu-ENK ( 10-12-10~‘o M) [Fig. 2(h-j)], and GAL ( 10~12-10-‘o M) [Fig. 2(k-m)]. The control response [Fig. 2(a)] in the absence of secretagogue is shown for comparison. The results show that ANP and Leu-ENK have secretagogue effects on the pancreas. PST at a lower concentration of lo-‘” M stimulated protein output whereas higher concentrations (lo-‘*-lo-” M) had no significant effect compared to control tissues. Similarly, galanin failed to evoke protein output. The basal amylase output in this series of experiment (15 animals) was 115 2 8.1 U ml-’ ( 100 mg tissue)-’ (n = 30). Figure 3 shows the effects of CCK-8 ( 10~‘2-10-‘o M) [Fig. 3(a)], PST (10-‘2-10-Lo IV) [Fig. 3(b)], ANP (lo-“-lo-” M) [Fig. 3(c)], and GAL (10-“-10~9 M [Fig. 3(d)] on amylase output from isolated pig pancreatic segments. CCK-8, PST, and ANP produced moderate increase in amylase output compared to GAL, which had no significant sescretagogue effect. Figure 4 shows the dose-dependent effect of Leu-ENK on amylase secretion in the absence [Fig. 4(b-e)] and in the presence [Fig. 4(g-i)] of 10m4 naloxone. The results show that Leu-ENK can elicit a dose-dependent increase in amylase output compared to the basal [Fig. 4(a)]. This increase was significantly (p < 0.05) antagonized by the endorphin receptor antagonist, naloxone [Fig. 4(f)].
Statistical Analysis All data provided are expressed as mean % SEM. Data were compared with Student’s t-test and only values with p < 0.05 were accepted as significant. Chemicals All reagents used in the measurement of protein output and amylase secretion were obtained from Sigma (UK). RESULTS
Immunohistochemistry ANP immunoreactivity was observed in nerve fibers and cells of the endocrine and exocrine pancreas [Fig. 1 (a)]. GAL-immunopositive cells were seen in close association with insulinpositive cells in the islets of Langerhans and in the exocrine pancreas [Fig. 1 (b)] . Leu-ENK was discernible in some of the insulin-positive cells of the islet of Langerhans and also in neurons of the exocrine pancreas [Fig. 1 (c-d)]. PST-immunoreactive cells were observed in the islet of Langerhans and in the wall of the ducts [Fig. 1 (e-f)].
DISCUSSlON
Immunohistochemistry Secretagogue-Evoked Secretion
Total Protein Output and Amylase
Employing the technique of double immunolabeling, we have demonstrated the presence and distribution of ANP-, PST-, LeuENK-, GAL-, and insulin-immunoreactive cells and fibers in the pig pancreas. The neuropeptides ANP, GAL, and Leu-ENK were found in close association with insulin-positive cells of the islet
The mean t SEM basal output of secretory protein from pig (15 animals) pancreatic segments was 0.35 + 0.01 mg ml-’ (100 mg tissue)-’ (n = 87). Figure 2 shows histograms of mean +
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showing the effects of varying concentrations (b-d) of atrial natriuretic peptide (ANP, (lo-‘lo-“M); (e-g) pancreas&&in (PST, (10-13-10-” M); (h-j) leucine-enkephalin (Leu-Enk, 10~‘2-10-“’ M), and (k-m) galanin (GAL, 10-‘2-10-‘o M) on protein output from pig pancreatic segments. The basal protein secretion in the absence of secretagogues shown for comparison (a). Each point is mean ? SEM. The n values in each histograms are the number of assays from 15 animals.
IMMUNOHISTOCHEMICAL
0
IDENTIFICATION
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FIG. 3. Histograms showing the effect of lo-“lo-“‘M CCK-8 (a), 10-‘2-10-ro M PST (b), lo-“lo-” M ANP (c), and lo-“low9 M GAL (d) on amylase secretion from isolated pig pancreatic segments. Values are expressed as percentage of the control basal level [115 ? 8.0 U ml-r (100 mg tissue)-‘] where number of assays equals to 30. Each point is mean ‘_ SEM. n = 15 animals.
of Langerhans. These neuropeptides were also distributed in the exocrine part of the pancreas. Leu-ENK was observed to colocalize with insulin in this study. The may indicate a role for LeuENK in the metabolism of insulin. PST, on the other hand, is produced and/or stored in islet and ductal cells. The localization of the PST in the islet of ILangerhans corroborates previous findings ( 18). PST was shown to be colocalized in the endocrine pancreas with most of the. islet hormones including, insulin, glucagon, and somatostatin (18). The distribution of ANP has hardly been studied in tmhepancreas and it is thus difficult to compare the results obtained here with that of the literature.
Among the few ANP-related peptides identified in the pancreas is a 126-amino acid prohormone (32). Similar immunohistochemical studies have identified GAL-immunoreactive nerve fibers near the acini, duct, and blood vessels of the pancreas of a number of species including the pig (22)) mouse and rat (6,2 1) , chicken ( 13), and man (29). Enkephalin-related peptides (19,28) have previously been shown to be present in the nerves of the rat and guinea pig pancreas. It is noteworthy that with the exception of GAL, the immunohistochemical identification of these peptides has not been studied extensively in the vertebrate and nonvertebrate pancreas. The association of these peptide-
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FIG. 4. Histograms showing the effect of different concentrations (10-‘2-10-‘o M) of Leu-ENK on amylase secretion in the absence (b-e) and presence of 10m4 naloxone (gj). The control (con) responses for KH (a) and naloxone (f) are shown for comparison. Values are expressed as percentage relative to the basal level. Each point is mean 2 SEM. n = 10 animals.
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containing cells and fibers with the exocrine and endocrine secretory structures of the pancreas suggests that they might be important neuromodulators of secretion. Indeed, the experiments involving protein output and amylase secretion indicate that LeuENK and ANP possess secretagogue function. It has been reported previously that the neuropeptides may possess neuroregulatory roles in controlling either blood flow, neurotransmitter release, or regulating secretagogue-evoked pancreatic secretion (9,12). A few of the several peptides that have also been identified in the pancreas of different species in previous studies include VIP, substance P, CGRP, and NPY ( 19,28,30).
Secretagogue-Evoked Total Protein Output and Amylase Secretion The study has also attempted to ascertain a functional role for the ANP, PST, Leu-ENK, and GAL identified in the pig pancreas. Compared to CCK-8, both ANP and Leu-ENK can also exert marked dose-dependent secretagogue effects on the pancreas. Moreover, the secretory action of Leu-ENK was blocked by naloxone, the endorphin antagonist, indicating the presence of Leu-ENK receptors on pancreatic acinar cells. The dose-dependent nature of ANP also indicates that ANP receptors are present on pancreatic acinar cells. In contrast, GAL had no significant effect on either total protein output or amylase secretion. On the other hand, PST only stimulated protein secretion at lo-l3 M, whereas higher concentrations had no significant effect compared to the control value. In contrast to its effect on protein output, PST evoked clear and detectable increases in amylase secretion at all concentrations tested in this study. Because GAL is present in the nerves of the pancreas but has no secretagogue effect in the pancreas, it is tempting to suggest that this neuropeptide may possess another modulatory role in the control of pancreatic function. The results have demonstrated that the peptides exerted differential effects on total protein output and amylase secretion in the pig pancreas. For
ET AL.
example, at lower concentrations the secretagogue peptides were more potent in releasing secretory proteins compared to higher concentrations. This observation would suggest that the results are not artifactual due to a measurement of the secretagogue peptide. Moreover, control segments have shown that the peptides had no detectable effect on the protein assay procedures. The question that now arises is whether the secretory proteins are released either from the acinar cells or islet cells in the pancreas following application of the secretagogue peptides. The results of the amylase experiments show that ANP, LeuENK, and PST can evoke moderate increases in amylase secretion compared to the gut hormone, CCK-8, indicating that these peptides are acting directly on acinar cells to elicit their secretagogue effect. It is noteworthy that in the in vivo dog preparation Leu-ENK has been shown to inhibit exocrine pancreatic secretion (17) whereas in the islet of Langerhans it evoked a moderate release of islet hormones ( 11). On the other hand, PST has been shown to inhibit the basal as well as the secretagogue-evoked pancreatic secretion in both the in vivo rat preparation (9) and in vitro guinea pig pancreas (15). Taken together, the evidence supports a role for these neuropeptides in the pancreas. However, further experiments are required to investigate precisely the cellular mechanism of action of these neuropeptides on either acinar cells or islet cells to elicit secretion. In conclusion, the study has demonstrated the presence and distribution of ANP, PST, Leu-ENK, and GAL in the pig pancreas, and some of these substances seem to have important neuromodulating roles in the control of pancreatic secretion. ACKNOWLEDGEMENTS This work was supported by the Wellcome Trust and the British Council. The authors wish to thank Dr. Sandra Child and Mr. Ashok Prasad (Media Production Unit, FMHS, United Arab Emirates University) for their valuable assistance in the production of the micrographs.
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10. Gorelick, F. S.; Jamieson, J. D.; Kinder, B.; Dobbins, J. Enkephalin inhibits pancreatic secretion. Clin. Res. 27:266 A; 1979. 11. Gorelick, F. S .; Jamieson, J. D. Structure-function relationship of the pancreas: In: Johnson, L. R., ed. Physiology of the gastrointestinal tract. vol. 2, 2nd ed. New York: Raven Press; 1987:1089-1098. 12. Herzig, K. H.; Dexter, S. L.; Tatemoto, K.; Owyang, C. Pancreastatin inhibits pancreatic enzyme secretion by presynaptic modulation of acetylcholine release. Am. J. Physiol. 262:G113-G117; 1992. 13. Hiramatsu, K.; Ohshima, K. Immunohistochemcal study on the distribution of galanin-containing nerves in the chicken pancreas. Histol. Pathohistol. 10:283-288; 1995. 14. Holst, J. J. Neuronal regulation of pancreatic exocrine function. In: Go, V. L. W.; Gardner, J. D.; Brooks, F. B.; Lebenthal, E.P.; Dimago, E. P.; Scheele, G. A., eds. The exocrine pancreas: Biology, pathobioloby and disease. New York: Raven Press; 1986:9-19. 15. Ishizuka, J.; Asada, I.; Posten, J.; et al. Effect of pancreastatin on pancreatic endocrine and exocrine secretion. Pancreas 4:277-281: 1989. 16. Kimura, W.; Jimi, A.; Miyasaka, K.; et al. Immunohistochemical study of the distribution of pancreastatin in endocrine tumours of the pancreas and in normal pancreatic tissue: Analysis of autopsy cases. Pancreas 6:688-693; 1991. 17. Konturek, S. J.; Tasler, J.; Geszkowski, M.; Jaworek, J.; Coy, D. M.; Schally, A. V. Inhibition of pancreatic secretion by enkephalin and morphine on dogs. Gastroenterology 74:851-855; 1978.
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18. Lamberts, R.; Schmidt, W. E.; Creutzfeld, W. Light and electron microscopical immunohistochemical localization of pancreastatin-like immunoreactivity in porcine tissues. H&chemistry 93:369-380; 1990. 19. Larsson, L. I. Innervation of the pancreas by substance P, enkephalin, vasoactive intestinal polypeptide and gastrin/CCK immunoreactive nerves. J. Histoclnem. Cytochem. 27:1283-1284; 1979. 20. Lenninger, S. The autonomic innervation of the exocrine pancreas. Med. Clin. North Am. 58:1311-1318; 1974. 21. Lindskog, S.; Ahren, B.; Dunning, B. E.; Sundler, F. Galanin-immunoreactive nerves in the mouse and rat pancreas. Cell Tissue Res. 264:363-369; 1991. 22. Messel, T.; Harling, H.; Bottcher, G.; Tohnsen, A. H.; Holst, J. J. Galanin in the porcine paacreas. Regul. Pept. 28: 161- 176; 1990. 23. Pearson, G. T.; Singh, .I; Daoud, M. S.; Davison, J. S.; Petersen, 0. H. Control of pancreatic cyclic nucleotide levels and amylase secretion by noncholinergic, nonadrenergic nerves. J. Biol. Chem. 256:11025-11031; 1981. 24. Pearson, G. T.; Singh, J.; Petersen, 0. H. Adrenergic nervous control of CAMP-mediated amylase secretion in the rat pancreas. Am. J. Physiol. 246:G563-G57.3; 1984. 25. Richens, C. A. The innervation of the pancreas. J. Comp. Neurol. 82:223-236; 1945. 26. Rindemecht, J.; Marbach, E. P. A new automated method for the determination of serum cu-amylase.Clin. Chim. Acta 29:107-110; 1970.
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27. Schmidt, W. E.; Siegel, E. G.; Lamberts, R.; Gallwitz, B.; Creutzfeldt, W. Pancreas&&n: Molecular and immunocytochemical characterization of a novel peptide in porcine and human tissues. Endocrinology 123:1395-1404; 1988. 28. Schultzberg, M.; Hokfelt, T.; Nilsson, G.; et al. Distribution of peptide and catecholamine containing neurons in the gastrointestinal tract of rat and guinea pig : Immunohistochemical studies with antisera to substance P, VIP, enkephalin, somatostatin, gastrinlcholecystokinin, neurotensin and dopamine-beta-hydroxylase. Neuroscience 5:689-744; 1980. 29. Shimosegawa, T.; Moriizumi, S.; Koizumi, M.; Kashimura, J.; Yanaihara, N; Toyota, T. Immunohistochemical demonstration of galanin-like immunoreactive nerves in the human pancreas. Gastroenterology 102:263-271; 1992. 30. Su, H. C.; Bishop, A. E.; Power, R. F.; Hamada, Y. M.; Polak, M. Dual intrinsic and extrinsic origins of CGRP- and NPY-immunoreactive nerves of rat gut and pancreas. 3. Neurosci. 7:2674-2678; 1987. 31. Sundler, F.; Alumets, J.; Hakanson, R.; Fahrenkrug, J.; Schaffalitzky de Muckadell, 0. Peptidergic (VIP) nerves in the pancreas. Histochemistry 55:173-176; 1978. 32. Vesely, D. L.; Palmer, P. A.; Giordan, D. Atrial natriuretic factor prohormone peptides are present in a variety of tissue. Peptides 13:165-170; 1992.
PIISO196-9781(96)00011-3
ELSEVIER
Immunohistochemical Identification and Effects of Atria1 Natriuretic Peptide, Pancreastatin, Leucine-Enkephalin, and Galanin in the Porcine Pancreas ERNEST ADEGHATE, * ’ ZSOLT EMBER,-/- TIBOR DONATH,? AND JAIPAUL SINGH$
DAVID J. PALLOT”
*Department of Human Anatomy, Faculty of Medicine and Health Sciences, United Arab Emirates University, P. 0. Box 17466, Al-Ain, United Arab Emirates fist Department of Anatomy, Semmelweis University Medical School, Tfizolto’ u. 58, Budapest, Hungary $Department of Applied Biology, University of Central Lancashire, Preston, England Received 5 November 1995 ADEGHATE, E., 2s. EMBER, T. DONATH, D. J. PALLOT AND J. SINGH. Zmmunohisfochemical ident$cafion and effecfs of atria1 nut&retie peptide, pancreastatin, leucine-enkephalin, and galanin in the porcine pancreas. PEPTIDES 17(3) 503-509, 1996.-This study demonstrates the presence and distribution of atrial natriuretic peptide (ANP) pancreastatin (PST), leucineenkephalin (Leu-IINK), galanin (GAL), and insulin in the pig pancreas. The effects of PST, ANP, Leu-ENK, and GAL on protein and amylase secretion were also investigated to determine their functional role in the control of pancreatic secretion. PST-immunoreactive cells were observed in the islet of Langerhans and in the wall of the ducts. Leu-ENK-immunopositive cells were observed in both the endo- and exocrine pancreas. It is colocalized with insulin in the islet of Langerhans. ANP immunoreactivity was discernible in nerve fibers and cells of the exocrine pancreas. GAL-immunopositive cells were observed in close association with insulin-posiblve cells in the islets of Langerhans and in the exocrine pancreas. Stimulation of isolated pancreatic segments with either ANP or Leu-ENK resulted in increased protein secretion and amylase output. The Leu-ENK-evoked amylase secretion was antagonized by naloxone. Pancreastatin was effective at all concentrations, but low concentration had more marked secretory effects whereas GAL failed to evoke any significant increases in either protein or amylase secretion. The results of the study have demonstrated a close association of peptidergic fibers with the secretory cells of the pancreas. The nerve fibers can release peptides that in turn can stimulate protein and amylase secretion. Porcine pancreas Atria1 natriuretic
Immunohistochemistry peptide
Leucine-enkephalin
THE innervation of the pancreas is provided mainly by the parasympathetic nerves ( 14,I 9,26) and to a lesser extent by the sympathetic nerves (14,24) of the autonomic nervous system. The main sources of innervat.lon include the vagus and the splanchnic nerves ( 14). The sites of nerve termination vary from species to species but in general they seem to terminate near blood vessels, basal membrane of pancreatic acinar and ductal cells, and islet cells ( 3,11,14). Stimulation of the parasympathetic nerves results in the release of acetylcholine (ACh), which in turn activates cholinergic muscarinic receptors. This in turn leads to the metabolism of membrane-bound phosphatidylinositol 1,4 bisphosphate (PIP*), resulting in the elevation of diacylglycerol (DG), inositol t&phosphate (II?,), and inositol tetrakisphosphate (IP,) to elicit secretion (4,5,8). DG stimulates protein kinase C
’ Requests
for reprints
should be addressed
to Ernest Adeghate,
Insulin
Galanin
Pancreastatin
whereas IP3 and IP, mobilize cellular calcium (Ca”). On the other hand, stimulation of adrenergic nerves results in the release of noradrenaline, which activates mainly p-receptors, resulting in the metabolism of adenosine 3,5 cyclic monophosphate (cyclic AMP), which mediates secretion (24). In addition to parasympathetic and sympathetic innervation of the pancreas, there is much evidence for the presence of noncholinergic, nonadrenergic, or peptidergic nerves that contain vasoactive intestinal polypeptide (VIP) in several animal species including rat, cat, dog, pig, and man (14,23,26). VIP elicits pancreatic secretion via cyclic AMP metabolism (23). Apart from VIP, immunohistochemical studies have demonstrated the presence of numerous neuropeptides, including gas&in-releasing peptide (GRP) , substance P, cholecystokinin, neuropeptide Y, calcitonin gene-re-
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ADEGHATE ET AL
IMMUNOHISTOCHEMICAL
IDENTIFICATION
lated peptide (CGRP) , and enkephalin-related peptides, mainly in the pancreas of rat and guinea pig (2,14,19,28,30). This study was designed to identify the distribution of atrial natriuretic peptide (ANP), pancreastatin (PST), leucine-enkephalin (LeuENK) ,and galanin (GAL) in the pig pancreas, especially because the gland has a similar innervation pattern to man. In addition, the study also investigated the effects of exogenous applications of ANP, PST, Leu-ENK, and GAL on total protein output and amylase secretion in the isolated pig pancreas to ascertain their functional roles in the stimulus-secretion coupling process. The effect of the gut peptide, cholecystokinin octapeptide (CCK-8), on amylase secretion was also investigated for comparison. METHOD
Immunohistochemistry All experiments were performed on segments of freshly slaughtered pig pancreas. Tissues were collected immediately after killing the animals and fixed for 48 h in a mixture of 2% formaldehyde and 15% of saturated aqueous picric acid solution in 0.1 M phosphate-buffered saline (PBS ) . The specimens were later immersed overnight in 20% sucrose solution and were subsequently sectioned into 40-50-km-thick slices using a vibroslice equipment (Chapden Instruments, USA). The sections were subsequently incubated for 48 h at 4°C with monoclonal guinea pig anti-insulin serum. After several washings in PBS (pH 7.4), the sections were incubated for 1 h at room temperature in biotinylated anti-rabbit immunoglobulin and later in avidin-biotinperoxidase complex. Sites of immunoreactions were detected using diaminobenzidine (DAB) solution (44 mg 300 ml -’ ) of PBS containing 0.04 ml of H202 (0.03%) for 3-5 min. A brown precipitate was observed in the sites of the antigen. After the DAB reaction, which indicates insulin-positive sites, the specimens were washed in PBS and incubated either in rabbit anti-GAL, anti-Leu-ENK, anti-ANP, or anti -PST. Incubation and washing periods were the same as for the insulin immunohistochemistry given above. Sites of immunoreaction were detected using diaminobenzidine solution (44 mg 300 ml-‘) of PBS containing 0.4% nickel ammonium sulphate and 0.04 ml of H,Oz (0.03%) for 3-5 min. The sites of unmunoreaction gave a dark-blue color. In some experiments, DAB only was used to identify PST and Leu-ENK. In all rinses PBS containing 0.5% Triton X-100 was used. After staining, the sections were counterstained with haematoxylin, dried, dehydrated, and mounted in De-PeX. (Serva, Heidelberg, Germany ) Sources of the Antisera ANP, PST, Leu-ENK, and GAL antibodies and antigens were bought from Peninsula Laboratories (California, USA). Inert and link antisera and the detecting reagents (avidin-biotin-peroxidase complex, DAB) were all products of DAK0 (Copenhagen, Denmark).
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Immunohistochemical
Controls
Control experiments included: i) primary antisera preabsorbed with 10e6 M of the corresponding peptide; ii) omission of the primary antisera followed by avidin-biotin-peroxidase complex staining; iii) substitution of the primary antisera with normal swine serum. These control incubations were carried out on sections adjacent to those used in the normal avidin-biotin-peroxidase complex procedure. In all these control experiments, immunolabeling was not observed. Specificity of the Antisera PST antibody has been shown by Lamberts et al. ( 18) and by Schmidt et al. (27) to be specific for PST, with no significant cross-reactivity with other peptides. GAL and Leu-ENK antibodies have been demonstrated by Adeghate and Singh ( 1) and by Schultzberg et al. (30) to be specific for GAL and Leu-ENK, respectively, with Leu-ENK having a 29% cross-reactivity with dynorphin A. ANP antiserum has also been shown to be specific for ANP and to not cross-react with any peptide (Peninsula Laboratories, 1993-94 catalogue). Measurements
of Protein Output
Immediately after killing the animals, the pancreas was removed and placed in an ice-cold Krebs Hesenleit solution (KH) and transported to the laboratory. The composition of KH in (mM) is: NaCl, 118; KCl, 4.76; CaCl*, 2.56; NaHC03, 25; KH2P04, 2.2; MgClz, 1.2, and glucose 10. The pancreas was cut into small segments (3-5 mg) and washed three times with 50 ml oxygenated KH solution (pH 7.4) maintained at 37°C to discard excess protein due to cutting the tissue. Tissue aliquots (approximately 30-40 mg) were placed in 5 ml plastic tubes containing control oxygenated KH solution (volume = 3 ml) and test KH solution (volume = 3 ml) containing different concentrations ( 10-13, 10-l’ M) of either ANP, GAL, Leu-ENK, or PST. The test tubes were incubated at 37°C in a shaking water bath for 20 min. After the incubation period the tissues were removed immediately from each test tube, blotted, and weighed. The concentration of protein in the supematant was determined by spectrotluorimetry using the Bradford (7) method. Bovine serum abumin (Sigma, UK) was used as standard. Protein output was expressed as mg ml-’ ( 100 mg tissue)-‘. Measurement
of Amylase Secretion
Portions (30-40 mg) of the pig pancreas were placed into 3ml aliquots of oxygenated KH solution containing different concentrations of either CCK-8 ( 10-‘2-10-‘o M), PST (lo-‘*lo-” M), ANP ( 10~“-10-9 M), GAL ( 10-“-10-9 M), or Leu-ENK ( 10-“-10-9 M). In some experiments the tissues were pretreated with 1O-4 naloxone for 5 min followed by incubation with Leu-ENK ( 10-“-10-9 M). In control experiments, pancreatic segments were incubated with either KH solution alone or with 10m4 naloxone. Samples were incubated at 37°C and constantly agitated for 20 min. At the end of the in-
FIG. 1. Light micrographs of haematoxylin-counterstained sections of the porcine pancreas. (a) Atria1 natriuretic peptide-immunoreactive neuron (arrow head), and fibers (arrow) in the exocrine pancreas and insulin-positive cells (brown reaction product) in the islet of Langerhans (i). x210. (b) Galanin (GAL)-immunopositive neuron (arrow) in the exocrine pancreas. Some GAL-positive cells (arrow head) lie within the islet (i). Insulincontaining cells are stained brown in the islet. x312. (c) Leucine-enkephalin (Leu-ENK)-immunoreactive cells (arrows) are distributed all over the exocrine pancreas. Leu-ENK is colocalized with insulin in some endocrine cells (open arrow). Insulin-containing cells are stained brown in the islet of Langerhans (i). X3 12. (d) Leu-ENK-immunoreactive neuron (arrow) in the interacinar areas. a, acinar tissue. X312. (e) Pancreastatin (PST)immunopositive cell (arrow) in the wall of a pancreatic duct (pt). X312. (f) PST-positive cells (arrow) in the islet of Langerhans (i). ~312. (a-c) Immunoreactivity developed with diaminobenzidine (DAB) and DAB/nickel ammonium sulphate. (d-f) Immunoreactivity developed with DAB alone.
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cubation amylase amylase amylase
period the supematant was decanted off and assayed for content by an on-line fluorimetric method (26) with LXas a standard. The tissues were blotted and weighed and expressed as units (U) ml-’ (100 mg tissue)-‘.
ET AL.
SEM protein output from pancreatic segments incubated with eitherANP(10-‘3-10-” M) [Fig. Zfb-d)],PSTf 10-‘3-10~” M) [Fig. 2(e-g)], Leu-ENK ( 10-12-10~‘o M) [Fig. 2(h-j)], and GAL ( 10~12-10-‘o M) [Fig. 2(k-m)]. The control response [Fig. 2(a)] in the absence of secretagogue is shown for comparison. The results show that ANP and Leu-ENK have secretagogue effects on the pancreas. PST at a lower concentration of lo-‘” M stimulated protein output whereas higher concentrations (lo-‘*-lo-” M) had no significant effect compared to control tissues. Similarly, galanin failed to evoke protein output. The basal amylase output in this series of experiment (15 animals) was 115 2 8.1 U ml-’ ( 100 mg tissue)-’ (n = 30). Figure 3 shows the effects of CCK-8 ( 10~‘2-10-‘o M) [Fig. 3(a)], PST (10-‘2-10-Lo IV) [Fig. 3(b)], ANP (lo-“-lo-” M) [Fig. 3(c)], and GAL (10-“-10~9 M [Fig. 3(d)] on amylase output from isolated pig pancreatic segments. CCK-8, PST, and ANP produced moderate increase in amylase output compared to GAL, which had no significant sescretagogue effect. Figure 4 shows the dose-dependent effect of Leu-ENK on amylase secretion in the absence [Fig. 4(b-e)] and in the presence [Fig. 4(g-i)] of 10m4 naloxone. The results show that Leu-ENK can elicit a dose-dependent increase in amylase output compared to the basal [Fig. 4(a)]. This increase was significantly (p < 0.05) antagonized by the endorphin receptor antagonist, naloxone [Fig. 4(f)].
Statistical Analysis All data provided are expressed as mean % SEM. Data were compared with Student’s t-test and only values with p < 0.05 were accepted as significant. Chemicals All reagents used in the measurement of protein output and amylase secretion were obtained from Sigma (UK). RESULTS
Immunohistochemistry ANP immunoreactivity was observed in nerve fibers and cells of the endocrine and exocrine pancreas [Fig. 1 (a)]. GAL-immunopositive cells were seen in close association with insulinpositive cells in the islets of Langerhans and in the exocrine pancreas [Fig. 1 (b)] . Leu-ENK was discernible in some of the insulin-positive cells of the islet of Langerhans and also in neurons of the exocrine pancreas [Fig. 1 (c-d)]. PST-immunoreactive cells were observed in the islet of Langerhans and in the wall of the ducts [Fig. 1 (e-f)].
DISCUSSlON
Immunohistochemistry Secretagogue-Evoked Secretion
Total Protein Output and Amylase
Employing the technique of double immunolabeling, we have demonstrated the presence and distribution of ANP-, PST-, LeuENK-, GAL-, and insulin-immunoreactive cells and fibers in the pig pancreas. The neuropeptides ANP, GAL, and Leu-ENK were found in close association with insulin-positive cells of the islet
The mean t SEM basal output of secretory protein from pig (15 animals) pancreatic segments was 0.35 + 0.01 mg ml-’ (100 mg tissue)-’ (n = 87). Figure 2 shows histograms of mean +
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showing the effects of varying concentrations (b-d) of atrial natriuretic peptide (ANP, (lo-‘lo-“M); (e-g) pancreas&&in (PST, (10-13-10-” M); (h-j) leucine-enkephalin (Leu-Enk, 10~‘2-10-“’ M), and (k-m) galanin (GAL, 10-‘2-10-‘o M) on protein output from pig pancreatic segments. The basal protein secretion in the absence of secretagogues shown for comparison (a). Each point is mean ? SEM. The n values in each histograms are the number of assays from 15 animals.
IMMUNOHISTOCHEMICAL
0
IDENTIFICATION
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FIG. 3. Histograms showing the effect of lo-“lo-“‘M CCK-8 (a), 10-‘2-10-ro M PST (b), lo-“lo-” M ANP (c), and lo-“low9 M GAL (d) on amylase secretion from isolated pig pancreatic segments. Values are expressed as percentage of the control basal level [115 ? 8.0 U ml-r (100 mg tissue)-‘] where number of assays equals to 30. Each point is mean ‘_ SEM. n = 15 animals.
of Langerhans. These neuropeptides were also distributed in the exocrine part of the pancreas. Leu-ENK was observed to colocalize with insulin in this study. The may indicate a role for LeuENK in the metabolism of insulin. PST, on the other hand, is produced and/or stored in islet and ductal cells. The localization of the PST in the islet of ILangerhans corroborates previous findings ( 18). PST was shown to be colocalized in the endocrine pancreas with most of the. islet hormones including, insulin, glucagon, and somatostatin (18). The distribution of ANP has hardly been studied in tmhepancreas and it is thus difficult to compare the results obtained here with that of the literature.
Among the few ANP-related peptides identified in the pancreas is a 126-amino acid prohormone (32). Similar immunohistochemical studies have identified GAL-immunoreactive nerve fibers near the acini, duct, and blood vessels of the pancreas of a number of species including the pig (22)) mouse and rat (6,2 1) , chicken ( 13), and man (29). Enkephalin-related peptides (19,28) have previously been shown to be present in the nerves of the rat and guinea pig pancreas. It is noteworthy that with the exception of GAL, the immunohistochemical identification of these peptides has not been studied extensively in the vertebrate and nonvertebrate pancreas. The association of these peptide-
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FIG. 4. Histograms showing the effect of different concentrations (10-‘2-10-‘o M) of Leu-ENK on amylase secretion in the absence (b-e) and presence of 10m4 naloxone (gj). The control (con) responses for KH (a) and naloxone (f) are shown for comparison. Values are expressed as percentage relative to the basal level. Each point is mean 2 SEM. n = 10 animals.
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containing cells and fibers with the exocrine and endocrine secretory structures of the pancreas suggests that they might be important neuromodulators of secretion. Indeed, the experiments involving protein output and amylase secretion indicate that LeuENK and ANP possess secretagogue function. It has been reported previously that the neuropeptides may possess neuroregulatory roles in controlling either blood flow, neurotransmitter release, or regulating secretagogue-evoked pancreatic secretion (9,12). A few of the several peptides that have also been identified in the pancreas of different species in previous studies include VIP, substance P, CGRP, and NPY ( 19,28,30).
Secretagogue-Evoked Total Protein Output and Amylase Secretion The study has also attempted to ascertain a functional role for the ANP, PST, Leu-ENK, and GAL identified in the pig pancreas. Compared to CCK-8, both ANP and Leu-ENK can also exert marked dose-dependent secretagogue effects on the pancreas. Moreover, the secretory action of Leu-ENK was blocked by naloxone, the endorphin antagonist, indicating the presence of Leu-ENK receptors on pancreatic acinar cells. The dose-dependent nature of ANP also indicates that ANP receptors are present on pancreatic acinar cells. In contrast, GAL had no significant effect on either total protein output or amylase secretion. On the other hand, PST only stimulated protein secretion at lo-l3 M, whereas higher concentrations had no significant effect compared to the control value. In contrast to its effect on protein output, PST evoked clear and detectable increases in amylase secretion at all concentrations tested in this study. Because GAL is present in the nerves of the pancreas but has no secretagogue effect in the pancreas, it is tempting to suggest that this neuropeptide may possess another modulatory role in the control of pancreatic function. The results have demonstrated that the peptides exerted differential effects on total protein output and amylase secretion in the pig pancreas. For
ET AL.
example, at lower concentrations the secretagogue peptides were more potent in releasing secretory proteins compared to higher concentrations. This observation would suggest that the results are not artifactual due to a measurement of the secretagogue peptide. Moreover, control segments have shown that the peptides had no detectable effect on the protein assay procedures. The question that now arises is whether the secretory proteins are released either from the acinar cells or islet cells in the pancreas following application of the secretagogue peptides. The results of the amylase experiments show that ANP, LeuENK, and PST can evoke moderate increases in amylase secretion compared to the gut hormone, CCK-8, indicating that these peptides are acting directly on acinar cells to elicit their secretagogue effect. It is noteworthy that in the in vivo dog preparation Leu-ENK has been shown to inhibit exocrine pancreatic secretion (17) whereas in the islet of Langerhans it evoked a moderate release of islet hormones ( 11). On the other hand, PST has been shown to inhibit the basal as well as the secretagogue-evoked pancreatic secretion in both the in vivo rat preparation (9) and in vitro guinea pig pancreas (15). Taken together, the evidence supports a role for these neuropeptides in the pancreas. However, further experiments are required to investigate precisely the cellular mechanism of action of these neuropeptides on either acinar cells or islet cells to elicit secretion. In conclusion, the study has demonstrated the presence and distribution of ANP, PST, Leu-ENK, and GAL in the pig pancreas, and some of these substances seem to have important neuromodulating roles in the control of pancreatic secretion. ACKNOWLEDGEMENTS This work was supported by the Wellcome Trust and the British Council. The authors wish to thank Dr. Sandra Child and Mr. Ashok Prasad (Media Production Unit, FMHS, United Arab Emirates University) for their valuable assistance in the production of the micrographs.
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18. Lamberts, R.; Schmidt, W. E.; Creutzfeld, W. Light and electron microscopical immunohistochemical localization of pancreastatin-like immunoreactivity in porcine tissues. H&chemistry 93:369-380; 1990. 19. Larsson, L. I. Innervation of the pancreas by substance P, enkephalin, vasoactive intestinal polypeptide and gastrin/CCK immunoreactive nerves. J. Histoclnem. Cytochem. 27:1283-1284; 1979. 20. Lenninger, S. The autonomic innervation of the exocrine pancreas. Med. Clin. North Am. 58:1311-1318; 1974. 21. Lindskog, S.; Ahren, B.; Dunning, B. E.; Sundler, F. Galanin-immunoreactive nerves in the mouse and rat pancreas. Cell Tissue Res. 264:363-369; 1991. 22. Messel, T.; Harling, H.; Bottcher, G.; Tohnsen, A. H.; Holst, J. J. Galanin in the porcine paacreas. Regul. Pept. 28: 161- 176; 1990. 23. Pearson, G. T.; Singh, .I; Daoud, M. S.; Davison, J. S.; Petersen, 0. H. Control of pancreatic cyclic nucleotide levels and amylase secretion by noncholinergic, nonadrenergic nerves. J. Biol. Chem. 256:11025-11031; 1981. 24. Pearson, G. T.; Singh, J.; Petersen, 0. H. Adrenergic nervous control of CAMP-mediated amylase secretion in the rat pancreas. Am. J. Physiol. 246:G563-G57.3; 1984. 25. Richens, C. A. The innervation of the pancreas. J. Comp. Neurol. 82:223-236; 1945. 26. Rindemecht, J.; Marbach, E. P. A new automated method for the determination of serum cu-amylase.Clin. Chim. Acta 29:107-110; 1970.
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