Functional control of chromogranin A and B concentrations in the body of the rat stomach

Regulatory Peptides, 40 (1992) 51-61

51

© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-0115/92/$05.00

REGPEP 01193

Functional control of chromogranin A and B concentrations in the body of the rat stomach Allan Watkinson and Graham J. Dockray MR C Secretory Control Group, Physiological Laboratory, University of Liverpool, Liverpool (UK) (Received 15 November I991; revised version received 9 March 1992; accepted 30 March 1992)

Key words: Chromogranin A; Chromogranin B; Omeprazole; Gastric fundus; Enterochromaffin-like cell; Food

Summary The chromogranins are soluble, acidic, proteins which are frequently co-stored in neuroendocrine cells with biogenic amines. In the gastric mucosa chromogranin A is localized to enterochromaffin-like cells which are the main source of histamine, and which are known to be regulated by circulating gastrin. We have used radioimmunoassays selective for the extreme C-terminal regions of chromogranin A and B to examine changes in gastric extracts following modulation of the gastric luminal contents. There were decreased concentrations of the two chromogranins in tissue extracts of rats after food withdrawal (which lowered plasma gastrin concentrations); inhibition of acid secretion with the H +/K +-ATPase inhibitor, omeprazole (which increased plasma gastrin concentrations) raised chromogranin A and B concentrations both in fasted rats, and in rats fed ad libitum. There was no evidence for altered patterns of posttranslational cleavage of chromogranin A or B with these treatments. The data indicate that chromogranin A and B concentrations in gastric ECL cells are regulated in parallel with histamine production, and are consistent with the idea that the chromogranins play a role in the formation and stabilization of the secretory granule involved in amine storage.

Correspondence to: G.J. Dockray, The Physiological Laboratory, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, UK.

52 Introduction

The three members of the chromogranin family, chromogranin A (CgA), chromogranin B (CgB) and secretogranin II, are widely distributed in neuroendocrine cells, and in recent years have attracted attention as markers for secretory cell function and pathology [ 1,2]. The physiological roles of these proteins are still incompletely understood, although it is clear that they are (a) putative precursors for several biologically active peptides, e.g., pancreastatin and chromostatin, which are active fragments of chromogranin A [3,4], and (b) potentially important as condensing agents in the formation of secretory granules [5]. In addition, it has been thought for many years that chromogranins might in some way act within the granule core to stabilise the storage of other secretory granule constituents, e.g., biogenic amines and ATP [6]. For the most part the relevant information comes from studies on the catecholamine-producing cells of the adrenal medulla, and it is not clear whether similar considerations apply to other cells expressing the chromogranin genes. Because chromogranin A is also found in gastric enterochromaffin-like (ECL) cells which produce histamine [7-11 ], these cells provide an alternative system in which to examine chromogranin functions. In the rat, ECL cells constitute the major endocrine cell population in the acid secreting mucosa of the stomach. The histamine that is released by these cells plays a central role in the control of acid secretion. Over many years an impressive body of evidence has been gathered to indicate that the acid-stimulating hormone gastrin is an important regulator of the rat ECL cell. Thus, gastrin release histamine, increases the activity of histidine decarboxylase ( H D C - which converts histidine to histamine), increases HDC mRNA abundance, and in the long term stimulates ECL cell proliferation [12-16]. If the chromogranins are functionally important constituents of the secretory granule core in these cells one would expect their levels to change in circumstances where histamine synthesis is known to be altered. In the present study we have examined this question by characterization of chromogranin in circumstances (fasting, and inhibition of acid secretion with the H +/K + -ATPase inhibitor omeprazole) that have reproducible effects on plasma gastrin concentrations [ 14,17 ]. The chromogranins were estimated by two new radioimmunoassays (RIA) using antibodies directed at the extreme C-terminal nonapeptide sequence of CgA (i.e., residues 440-448) and CgB (i.e., residues 647-650).

Materials and Methods

Peptide synthesis and antibody production. The peptides YIAEKFSQRG (rat[Y° ]CGB649_655), YHQLQALRRG (rat[Y°]CgA440_448), YKKEEEGSAN (rat[Y°]CgA409_417), YLAKRAMENM (rat[Y°]secretograninlIssl_sS9 and YKVAHQLQAL (rat[Y°]CgAa3v_445) were synthesized by standard solid phase methods using fmoc chemistry [18-20]; L-amino acids were used throughout except for the N-terminal tyrosines which were in the D-configuration; these residues were included to facilitate iodination of the peptides for use in RIA, and to stabilize against aminopeptidase activity. The crude synthetic peptides were purified by reverse phase

53

HPLC on Ultrasphere C8 semipreparative column with acetonitrile containing 0.1 ~o (v/v) trifluoroacetic acid (TFA). Antibodies to CGA440_448and CGB649_655were obtained by immunization of rabbits with peptides coupled to porcine thyroglobulin with glutaraldehyde as previously described [21]; the incorporation of peptide into conjugate was monitored using radiolabelled peptide. Rabbits (n = 4 or 5, for each peptide) were immunized with the equivalent of 20-50 nmol of peptide emulsified in Freund's complete adjuvant and boosted at 6 week intervals. The highest titre antibodies were used for RIA. Radioimmunoassay. C-terminal CgA-immunoreactivity (CgA440_a48-ir, i.e., YHQLQALRRG-ir) was determined by RIA using antiserum L346 at a dilution of 1:15,000 in 1 ml of phosphate buffer (pH 7.4) containing 1.0~o (v/v) Bovumin (Ortho Diagnostics), 50 mM sodium chloride, 1 mM polybrene, 2 mM EDTA and 0.05~o (w/v) sodium azide at 4 °C for 2-3 days. C-terminal CgB-immunoreactivity (CgB649_ 655-ir, i.e., YIAEKFSQRG-ir) was determined using antiserum L338 at a dilution of 1:70,000 in l ml of phosphate buffer (pH 7.4) containing 0.5~o (v/v) Bovumin for 2 days at 4 ° C. The synthetic peptides were radiolabelled by the chloramine T method and purified by reverse phase HPLC as previously described [21 ]. Antibody bound and free radiolabelled peptides were separated by centrifugation with a suspension of charcoal/dextran/non-fat milk powder/water, in the ratio 10:1:1:100 (w/v) (100 #1) for L346 and 10:1:2:100 (100 #1) for L338. Quantification of unknown samples was made by reference to standards of the respective synthetic peptide. The concentration of YHQLQALRRG required to inhibit binding of label to L346 by 50~o was 255 + 29 pM (mean + S.E., n = 10) and the concentration of YIAEKFSQRG that inhibited binding of label to L338 was 22.4 + 1.3 pM (n = 10) (Fig. 1). Antibody specificity was determined by comparison of the potencies of a range of synthetic peptides in inhibiting binding of label to antibody; in addition studies were performed of the immunochemical potency of native chromogranin A isolated from bovine adrenal chromaffin cells [21], and of synthetic peptides modified by digestion with trypsin, and carboxypeptidases A or B.

100

100

75

75

50

50

25

25

II1

¢= ~t

r

.

~

~

0.5

5

50

500

Concentration

(pM}

0

5

50

500

5000

Concentration (pM)

Fig. 1. lnh b t on o" bind lg of radiolabcllcd CgB¢,47-6ss to antibody L338, and of radiolabclled Q'gA44o_448 to L346 (right) by graded concentrations of the respective standard peptide (see also Table I).

54

Tissue extraction. Wistar rats (female) were fed ad libitum or fasted on wire bottomed cages for 48 h (water ad libitum). Both fed and fasted rats received either vehicle (0.25 ~o (w/v) methyl cellulose) or omeprazole (H~tssle, G0teborg) in a dose of 400 #mol.kg- 1 in 0.25 ~o methyl cellulose by gavage daily. Rats were decapitated and trunk blood collected into heparinized tubes for the assay of plasma gastrin by published methods [22]. The acid-secreting part of the stomach was removed, chopped into small pieces and immediately frozen on dry ice. The tissue pieces were extracted by boiling in water (0.1 g/ml) for 10 rain followed by homogenization. Insoluble material was removed by centrifugation at 5000 x g for 15 min and the resultant supernatant was defatted with 0.5 volumes of dichloromethane and centrifuged at 13,000 x g for 15 min. The aqueous layer was removed, leaving behind the precipitate at the interface, and aliquots were lyophilized for further analysis. When the insoluble material left after boiling water extraction was re-extracted with 3 ~o acetic acid no further immunoreactivity was observed, indicating that CgA and B peptides were recovered at neutral pH. Analytical Sephadex G50 gelfiltration. Lyophilized extracts were reconstituted in water and insoluble material removed by centrifugation. They were fractionated on Sephadex G50 superfine (1 x 100 cm) eluted with 0.05 M ammonium bicarbonate and 0.05% (w/v) sodium azide at 4°C. Fractions of 1.0 ml were collected; aliquots were lyophilized for determination of chromogranin immunoreactivity by RIA. The void and bed volumes were determined using Dextran blue and NaX25I, respectively. Statistics. Results are expressed as mean + S.E.; comparisons between groups were made by Student's t-test and were considered significant when P<0.05. Results

Antibody characterization Antibody L346 showed specificity for the intact C-terminus of CgA. In particular the synthetic C-terminal nonapeptide analogue of CgA, but not a fragment lacking the last three residues, inhibited binding of label (Table I). Moreover, digestion of YHQLQAL-

TABLEI Re~ativep~tencies~fchr~m~granin-derivedpeptidesininhibitingbinding~f~abe~t~antib~dy ~ L346 YHQLQALRRG Bovine CgA YIAEKFSQRG YIAKRMENM YKVAHQLQAL Y K K E E E G SAN

< < < <

1.0 0.091 0.0001 0.00001 0.0001 0.0001

L338 < 0.0001 < 0.0001 1.00 < 0.0001 < 0.0001 < 0.0001

~ The standard peptide is assigned a potency of 1.00 in each case; the immunochemical potencies of the other peptides are expressed as the ratio of concentration of standard and test peptide required to inhibit binding of label by 50%.

55 R R G with carboxypeptidase A (CPA: 10 U for 1 h at 37°C) reduced the immunoreactivity by 8 8 0 , and digestion with trypsin (10 #g for 3 h at 37°C) decreased immunoreactivity by 90~o. In contrast carboxypeptidase B digestion (CPB) (0.2 pg for 1 h at 37 ° C) had no effect, either before or after trypsinization. The C-terminal nonapeptide analogues of secretogranin II and CgB showed no activity in this assay, and neither did [Y°]CgA409_417 and [Y°]CgA333_34o (Table I). Purified bovine CgA, which has a C-terminal region differing in two amino acid substitutions compared to the rat peptide, had approx. 10-fold lower potency than the synthetic rat peptide. Antiserum L338, raised to the synthetic C-terminal nonapeptide of CgB showed high specificity for this sequence. The corresponding C-terminal fragments of rat CgA, and secretogranin II or bovine CgA itself, did not inhibit binding of label to this antiserum (Table I). Digestion with trypsin of Y I A E K F S Q R Q (10 #g for 3 h at 37°C) totally abolished immunoreactivity, similarly digestion with carboxypeptidase A (10 U for 1 h at 37°C) abolished immunoreactivity, but digestion with carboxypeptidase B had no effect. Rat CgA fragments [Y°]CgA437_445, [Y°]CgA4o9_417 and [Y°]CgA448_456 did inhibit binding of label to antiserum at 10/~M. Gastric corpus chromogranin A and B In rats fasted for 48 h the plasma gastrin concentrations (13.6 + 1.2 pM; n = 20) were significantly reduced (P<0.05) compared with rats fed ad libitum (35.4_+ 3.5 pM). Treatment of either fasted or fed rats with omeprazole produced a marked increase in plasma gastrin (116 + 35 pM; n = 24 and 283 + 31 pM, n = 14, respectively). In rats fasted for 48 h there was a small but significant (P<0.05) decrease in the concentration of CgA-ir measured with antibody L346 compared with animals fed ad libitum (Fig. 2). When omeprazole was administered to the fasted animals the decrease in CgA levels was prevented and when administered to fed animals there was a significant increase in tissue CgA concentration ( P < 0.01) compared with control rats fed ad libitum. The concentrations of CgB in rat corpus were lower than those of CgA, but the effects of fasting and omeprazole treatment were more pronounced (Fig. 3).

80 0

E

O.

I

0

40

t12

._= I

o

C

Omep

FAST.

C

Omep

FED

Fig. 2. Concentrations of chromograninA measured by antibody L346 in boiling water extracts of corpus of rats fasted for 48 h (Fast C, n = 20), fasted and treated with omeprazole(Fast Omep;n = 24), fed ad libitum (Fed C; n = 20), or fed ad libitum and treated with omeprazole(Fed Omep; n = 14). Results are means + SE; *, significantlydifferent from control, P<0.05, **, P<0.01, t-test.

56

10 o E I ,.n

L)

II) 1

//

O C

Omep

C

FAST.

Ornep FEE)

Fig. 3. Concentrations of chromogranin B measured by antibody L338 in boiling water extracts of corpus from fasted for 48 h or fed ad libitum, and treated with omeprazole. *Significantly different from control P<0.001. See Fig. 2 for further details.

Fasting for 48 h markedly reduced CgB-ir (P< 0.001), whereas the treatment of fasted animals with omeprazole produced a substantial elevation of CgB tissue concentrations compared with fed control animals (P<0.001). Omeprazole treatment of rats fed ad libitum markedly increased tissue CgB concentrations compared with control rats (P<0.001). To characterize the immunoreactive forms involved in the omperazole-induced increase in CgA-ir and CgB-ir, extracts from each experimental group were fractionated by analytical Sephadex G50 gel filtration. The CgA-ir fractionated into three peaks: high molecular weight material that emerged in the void region (Kay = 0), material with an apparent molecular weight of 4 kDa (Kay= 0.4) and material with an apparent molecular weight of 1.5 kDa (Kay = 0.85) (Fig. 4). No difference in the relative proportion of these peaks was found in the different experimental groups. CgB-ir predominantly emerged in the void region of Sephadex G50 (Kav= 0), but there was a small peak of low molecular weight material (Kay = 0.9) (Fig. 5A and B). In contrast to the

m

O 0 0

0.25

0.5

0.75

1

Kay Fig. 4. Separation on Sephadex G50 of extracts of rat corpus assayed with antibody L346. Columns (1 x 100 cm) were eluted with ammonium bicarbonate (0.05) and fractions of 1.0 ml collected for assay. Void and total column volumes were determined by Dextran Blue and Na125I.

57

0.5

, o~

A.

0.25

o

.,I

o 1.o

,---

,-

,

-

_

,

B

(9

E I

o

o.s

0

0.25

O.SO

0.75

1.00

Ka~

Fig. 5. Separation on Sephadex G50 of extracts of rat corpus assayed with L338. See Fig. 3 for further details. (A) Results from animals fasted for 48 h; open symbols show the elution profile from an omeprazoletreated rat, and closed symbols from a control rat. (B) Results from animals fed ad libitum; symbols as in A.

gel filtration chromatographs of the CgA- derived peptides, omeprazole treatment, in fasted and fed animals, clearly increased the void region CgB immunoreactive material, compared with their respective controls. This increase appeared to include a 'shoulder' of immunoreactivity at K~v = 0.1.

Discussion The results of the present study provide evidence that the concentrations of CgA and CgB in the corpus of the rat stomach are regulated by the luminal contents of the stomach. Moreover, CgA and B concentrations change in parallel with plasma gastrin concentrations. It is well recognized that chromogranin A is localised to ECL cells of the gastric mucosa, and that these cells respond to gastrin by secretion of histamine and increases HDC activity and mRNA abundance [8-16]. The data therefore indicate that enhanced production of chromogranin is a further feature of the ECL cell response to gastrin. Surprisingly, changes in CgB concentrations were more dramatic than those of CgA. Previous studies have localised CgB to the G-cells of the pyloric antral mucosa but have not described this protein in the corpus mucosa. Our findings suggest that CgB expression is particularly sensitive to the presence of gastric acid (i.e., it was elevated after inhibition with omeprazole); regardless of the functional significance of CgB in the corpus, the present findings suggest this protein may be a useful

58

guide to achlorhydruia-induced changes in endocrine cell function in the corpus mucosa.

Current ideas of chromogranin function had developed in two different directions. Firstly, chromogranins have long been suspected to function in some way as stabilisers of the granule interior [6], More recently this idea has been extended by the suggestion that chromogranins may be important in the segregation of secretory proteins into regulatory secretory granules in the terminal Golgi region [5,23]. Secondly, there is evidence that these proteins are precursors of smaller biologically active molecules. Regulatory peptide precursors typically contain pairs of basic residues that are sites of intracellular cleavage in the production of the active molecular form. In CgA there are 10 such cleavage sites; and in CgB there are 20. Two fragments of CgA, pancreastatin and chromostatin, have been shown to possess biological activity and one of these is generated naturally by posttranslational processing of chromogranin in the pancreas [3,4]. Recent studies have shown that different patterns of posttranslationai processing of CgA are discernable in different populations of endocrine cells in the gut, pancreas and adrenal [21,24,25]. In the bovine antrum chromogranin A was processed to pancreastatin but in the acid secreting mucosa this processing step did not occur [21]. In the present study, we found that high molecular weight forms of CgA and CgB immunoreactivity predominated in rat acid-secreting mucosa; in other experiments using SDS page electrophoresis and immunoblotting we identified major bands corresponding to intact CgA and CgB in all groups of rats. Our radioimmunoassays for both proteins use antibodies reacting at the extreme C-terminus of the chromogranin molecule. These antibodies do not react with peptides generated by cleavage in the C-terminal region, but beyond this we would expect them to react with all fragments and forms of CgA or B containing an intact C-terminus. In the present context, it is important to note that there were no obvious changes in posttranslational processing of CgA with differing patterns of stimulation or inhibition of ECL cells. A further assay system that reads an internal sequences (409-417) of CgA, generated by cleavage at the second pair of basic residues from the C-terminus of CgA, indicated concentrations about 5 °/o those of intact CgA and these were similar in the different experimental groups. Taken together the evidence suggests that modulation of posttranslational processing of CgA does not occur in the present experimental circumstances. The possible intragranular functions of chromogranin are of interest in view of the attention that has been given to the significance of complexes between histamine and other granule contents in the mast cell [26,27]. Whether or not histamine and chromogranin A form a stable complex in ECL cells has not yet been studied in detail. The demonstration that chromogranin levels change in circumstances that alter histamine production suggests, however, that mechanisms are established in ECL cells to maintain a balance between the different components of such a complex. It is now clear that the luminal contents of the stomach determine many aspects of gastric endocrine cell function. It has been clear for many years that food (particularly protein) releases gastrin and that acid in the lumen inhibits gastrin release (see Ref. 28). It is now generally thought that acid acts via the release of somatostatin which in turn is a paracrine inhibitor of gastrin secretion. Suppression of acid secretion by the H +/K + -ATPase inhibitor omeprazole produces a marked increase in plasma gastrin.

59

In addition to changes in hormone secretion, it is now recognized that the same luminal factors (protein and acid) determine gastrin and somatostatin mRNA abundance, and in the case of gastrin also alter posttranslational processing [29-32]. Changes in plasma gastrin concentrations have consequences for ECL cell function that are out lined above. The present data add control of tissue chromogranin concentrations to the list of endocrine cell responses evoked by changes in the gastric luminal environment. It seems reasonable to suppose that the changes in chromogranin levels are secondary to altered synthesis which might be reflected in changed mRNA abundance, and we have preliminary evidence to suggest this might be the case (R. Dimaline, A. Sandvik and G.J. Dockray, unpublished observations). The prolonged achlorhydria produced by omeprazole-treatment for many months leads to an ECL cell dysplasia in the rat that eventually progresses to gastric carcinoid tumours [13,14]. This progression is thought to be rare or absent in man. The chromogranins are at present amongst the most valuable of markers for studying ECL cell numbers and distribution. The present data were obtained over relatively short periods of time when numbers of ECL cells would not be expected to change. The findings indicate, however, that in addition to changes that are a consequence of long term hypergastrinaemia there may also be relatively short term alterations in gastric chromogranin levels. The fact that chromogranin levels might be acutely modulated by the gastric luminal environment must therefore be kept in mind in interpreting studies on the distribution of chromogranin immunoreactivity in the stomach. More generally the present data suggest that chromogranin tissue concentrations are a further valuable way of monitoring ECL cell function.

Acknowledgements We are grateful to Dr. J. Smith (Department of Biochemistry, University of Liverpool) for synthesis of peptide, to Mrs. C. Carter for her help in preparing the manuscript, and to the MRC for financial support.

References 1 Simon, J.-P. and Aunis, D., Biochemistry of the chromogranin A protein family, Biochem. J., 262 (1989) 1-13. 2 Fischer-Colbrie, R., Hagn, C. and Schober, M., Chromogranins A, B and C: widespread constituents of secretory vesicles, Ann. N.Y. Acad.Sci., 493 (1987) 120-134. 3 Tatemoto, K., Efendic, S., Mutt, V., Makk, G., Feistner, G.J. and Barchas, J. D., Pancreastatin, a novel pancreatic peptide that inhibits insulin secretion, Nature 324 (1986) 476-478. 4 Galindo, E., Rill, A., Bader, M.-F. and Aunis, D., Chromostatin, a 20-amino acid peptide derived from chromogranin A, inhibits chromaffin cell secretion, Proc. Natl. Acad. Sci. USA, 88 (1991) 1426-1430. 5 Wiedenmann, B. and Huttner, W.B., Synaptophysin and chromogranins/secretogranins - widespread constituents of distinct types of neuroendocrine vesicles and new tools in tumor diagnosis, Virchows Archiv. Cell Pathol., 58 (1989) 95-121. 6 Helle, K. B., Chromogranins: universal proteins in secretory organelles from Paramecium to man, Neurochem. Int., 17 (1990) 165-175,

60

7 Hakanson, R., Bottcher, G., Ekblad, E., Panula, P., Simonssom M., Dohlsten, M., Hallberg, T. and Sundler, F., Histamine in endocrine cells in the stomach, Histochemistry, 86 (1986) 5-17. 8 Rindi, G., Buffa, R., Sessa, F., Tortora, O. and Solcia, E., Chromogranin A, B and C immunoreactivities of mammalian endocrine cells, Histochemistry, 85 (1986) 19-28. 9 Grube, D., Bargsten, G., Cetin, Y. and Yoshie, S., Chromogranins in mammalian GEP endocrine cells: their distribution and interrelations with co-stored amines and peptides, Arch. Histol. Cytol., 52 (1989) 91-98. 10 Cetin, Y., Muller-Koppel, L., Aunts, D., Bader, M-F. and Grube, D. Chromogranin A (CgA) in the gastro-entero-pancreatic (GEP) endocrine system, Histochemistry, 92 (1989) 265-275. 11 Cetin, Y. and Grube, D., hnmunoreactivities for chromogranins A and B, and secrctogranin II in the guinea pig entero-endocrine system: cellular distributions and intercellular heterogeneities, Cell Tiss. Res., 2643 (1991) 231-241. 12 Snyder, S.H. and Epps, L., Regulation of histidine decarboxylase in rat stomach by gastrin: the effect of inhibitors of protein synthesis, Mol. Pharinacol., 4 (1968) 187-195. 13 Larssom H., Carlsson, E., Mattsson, H., Lundell, L., Sundler, F., Sundell, G., Wallmark, B., Watanabe, T. and Hfikanson, R., Plasma gastrin and gastric enterochromaffinlike cell activation and proliferation, Gastroenterology, 9(I (1986) 391-399. 14 Larsson, H., Hhkanson, R., Mattsson, H., Ryberg, B., Sundler, F. and Carlsson, E., Omeprazole: its influence on gastric acid secretion, gastrin and ECL cells, Toxicol. Pathol., 16 (1988) 267-272. 15 Waldum, H.L. and Sandvik, A.K., Histaminc and the stomach, Scand. J. Gastroenterol., 24 (1989) 130-139. 16 Dimaline, R. and Sandvik, A. K., Histidine decarboxylase gene expression in rat fundus is regulated by gastrin, FEBS Lett., 281 (1991) 20-22. 17 Dimaline, R., Evans, D., Varro, A. and Dockray, G.J., Reversal by omeprazole of the depression of gastrin cell function by fasting in the rat, J. Physiol., 433 (1991) 483-493. 18 Benedum, U. M., kamouroux, A., Konecki, D. S., Rosa, P., Hille, A., Baeuerle, P. A., Frank, R., Lottspeich, F., Mallet, J. and Huttner, W. B., The primary structure of human secretogranin I (chromogranin B): comparison with chromogranin A reveals homologous terminal domains and a large intervening variable region, EMBO J., 6 (1987) 1203-1211. 19 Iacangelo, A., Okayama, H. and Eiden, L. E., Primary structure of rat chromogranin A and distribution of its mRNA, FEBS Left., 227 (1988) 115-121. 20 Forss-Petter, S., Danielson, P., Battenberg, E., Bloom, F. and Sutcliffe, J. G., Nucleotide sequence and cellular distribution of rat chromogranin B (Secretogranin l) mRNA in the rat neuroendocrine system, J. Mol. Neurosci., 1 (1989) 63-75. 21 Watkinson, A., Jonsson, A-C., Davison. M., Young, J., Lee, C.M., Moore, S. and Dockray, G.J.. Heterogeneity of chromogranin A-derived peptides in bovine gut, pancreas and adrenal medulla, Biochem. J.. 276 (1991)471-479. 22 Dockray, G. J., Best, L. and Taylor. I. L., hnnmnochemical characterization ofgastrin in pancreatic islets of normal and genetically obese mice, J. Endocrinol., 72 (1977) 143-151. 23 Rosa, P., Weiss, U., Peppcrkopk, R., Ansorge, W., Nichrs, C., Stelzcr, E. H. K. and Huttner, W. B., An antibod> against secretogranin I (chromogranin B) is packaged into secretory granules, J. Cell Biol., 109 (1989) 17-34, 24 Curry, W.J., Johnston, C. F., Shaw, C. and Buchanan, K.D., Distribution, and partial characterization of immunoreactivit~ to the putative C-terminus of rat pancreastatin, Regul. Pept., 30 (1990) 207-219. 25 Curry, W.J., Johnston. C.J., Hutton, J.C., Arden, S.D., RutherR~rd. N.G., Shaw, C. and Buchanan, K.D., The tissue distribution of rat chromogranin A-derived peptidcs: evidence for differential tissue processing front sequence specific antisera, Histochemistry, 96 (1991) 531-538. 26 Thon, I. L. and Uvnas, B., Modes of storage of histamine in mast cells, Acta PIDsiol. Scand., 67 (1966) 455-47(I. 27 Uvnas, B., Aborg, C.-H. and Bergendorff, A., Storage of histamine in mast cells. Evidence for an ionic binding of histamine to protein carboxyls in the granule heparin-protein complex, Acta Physiol. Scand. Suppl. 336 (1970) 1-26. 28 Dockray G.J. and Gregory, R.A., Gastrin, In G.M. Makhlouf (Ed.), Handbook of Physiology Gastrointestinal system, American Physiological Society, Bethesda, 1989, pp. 311-336.

61 29 Brand, S.J. and Stone, D., Reciprocal regulation of antral and somatostatin gene expression in omeprazole-induced achlorhydria, J. Clin. Invest., 82 (1988) 1059-1066. 30 Wu, S.V., Giraud, A., Mogard, M., Sumii, K. and Walsh, J.H., Effects of inhibition of gastric secretion on antral gastrin and somatostatin gene expression in rats, Am. J. Physiol., 258 (1990) G788-G793. 31 Wu, V.S., Sumii, K., Tari, A., Mogard, M. and Walsh, J.H., Regulation of gastrin somatostatin gene expression, Metabolism, 39 suppl. 2 (1990) 125-130. 32 Varro, A., Nemeth, J., Bridson, J., Lee, C., Moore, S. and Dockray, G.J., Processing of the gastrin precursor: modulation of phosphorylated, sulfated and amidated products, J. Biol. Chem., 265 (1990) 21476-21481.