Acta Physiol Scand 1990, 138, 369-376

Binding of cholecystokinin and somatostatin t o isolated porcine gastric mucosal cells and effects on aminopyrine uptake L. S J O D I N , L. J. E N G L U N D and S. MARDH* Pharmacological Division, Department of Drugs, National Board of Health and Welfare, and * Department of Medical and Physiological Chemistry, Uppsala University, Sweden

SJOOIN,L., ENGLUNO,L. J. & M ~ R D HS., 1990. Binding of cholecystokinin and somatostatin to isolated porcine gastric mucosal cells and effects on aminopyrine uptake. Acta Ph.ysiol Srand 138,369-376. Received 6 February 1989, accepted 31 October 1989. ISSN 0001-6772. Pharmacological Division, Department of Drugs, National Board of Health and Welfare, and Department of Medical and Physiological Chemistry, Uppsala University, Sweden. Mucosal cells were prepared by enzymatic digestion of porcine gastric mucosa with pronase and collagenase. The resulting cell suspension contained 1-1 5 ohparietal cells, which responded to histamine stimulation by an up to zo-fold increase in [“Claminopyrine accumulation over control levels. Cholecystokinin-8 (CCK-8) evoked a more moderate stimulation of [%]aminopyrine accumulation, whereas somatostatin inhibited histamine-stimulated accumulation. Parietal cells were enriched by elutriation and isopycnic centrifugation on density gradients of Percoll. A fraction with 6096 parietal cells bound approximately three times more iodinated CCK-8 than a fraction containing 70% non-parietal cells. Binding of [‘251]BH-CCK-8 to preparations containing 3&60% parietal cells was specifically inhibited to about 50% by I O - ~M unlabelled CCK-8 but not by bombesin. Cell fractions containing about 30 yo parietal cells also bound [‘251]somatostatin.Unlabelled somatostatin at I O - ~M inhibited tracer binding by about 50%, while CCK-8 did not affect somatostatin binding to such a preparation. The results suggest the existence of specific receptors for CCK and somatostatin on porcine parietal cells exerting a regulatory influence on acid secretion. Key m o d s ; acid production, aminopyrine, cholecystokinin, parietal cell, receptor, somatostatin. T h e gastric acid secretory glands are regulated in vivo by complex interactions between nervous and hormonal agents (Sjodin 197za). Some of the observed effects on acid secretion may well be indirect by secondary release of hormones, neurotransmitters or paracrine kctors. Thus, it has been suggested that gastrin and other stimulants of acid secretion act through release of histamine (Code 1965, Bergqvist & Obrink 1979). An important method of distinguishing between direct and indirect effects is to study the actions of the regulatory agents on isolated parietal cells. Correspondence : Lars Sjodin, Socialstyrelsens Lakemedelsavdelning, PO Box 607, S-751 25 Uppsala, Sweden.

A number of studies of acid secretory mechanisms have been performed with more or less pure preparations of parietal cells (Soll 1980, Batzri & Dyer 1981, MHrdh et al. 1984, 1987). However, few studies have been undertaken in order to characterize hormonal binding sites on gastric cells (Reyl et al. 1979, Soll et al. 1984, Park et al. 1987). Cholecystokinin has been shown to both stimulate (Sjodin 1972 b, c) and inhibit (Gillespie & Grossman 1964, Johnson & Grossman 1970, Sjodin 197zd) canine gastric acid secretion depending on the prevailing experimental conditions. Somatostatin was found to be a potent inhibitor of gastrin-stimulated acid secretion (Bloom et al. 1974, Uvnas-Wallensten et al.

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1981). T h e s e effects might have been d u e t o direct interaction of the agent with parietal cells or by indirect mechanisms influencing the acid secretory glands. T h e present study was undertaken in order to measure binding of radioiodinated cholecystokinin and somatostatin t o preparations of gastric mucosal cells and to relate such binding to modulation of acid secretion, as determined b y ["Claminopyrine uptakc.

Fifty millilitres of modified HBSS with z mM E G T A were added and the incubation was continued for 10 min at 37 "C. T h e suspension was then transferred to centrifuge tubes and spun at 50 g for 5 min. T h e supernatant was aspirated and the pellets were washed twice with 25 ml modified HBSS. T h e mucosal pieces were then digested with ~ o o m lof medium with addition of 0.9 mg ml-' collagenase and I % BSA for j j min during continuous magnetic stirring under a flow of oxygen. Thereafter, the suspension was filtered successively through nylon nets of zoo-, 75- and 25p m mesh. Cells were pelleted by centrifugation at 50 g for 5 min. After aspiration of supernatant, the pellet M A T E R I A L S AND METHODS was washed once and suspended in 25 ml of modified Porcine stomachs were obtained about zo min after HBSS with 106 BSA and, if not otherwise stated, slaughter at a local abbatoir. Collagenase (EC 0.5 mM Ca. The normal yield was 1 3 x IO* cells, of 3 . 4 . 2 4 . 3 ) and pronase (from S.grisezts) were from which 1-154, were parietal cells, counted as large finely granular cells in light microscopy. This prepBoehringer (Mannheim, FRG). Histamine-HC1, .%*', 0'-dibutyryl guanosine 3', 5' cyclic monophosphate aration is called mucosal cells. Isolation of parietal cells. I n certain experiments, (dibutyryl cyclic GMP, dbc GMP), soybean trypsin inhibitor, pepstatin '4, phenylmethylsulphonyl- parietal cells were enriched from the mucosal cell fluoride and phosphoramidon were from Sigma preparation by elutriation followed in some cases by (St Louis, MO, USA) and bovine serum albumin density gradient centrifugation as earlier described (fraction V) from Miles Lab., Elkhart, IN, USA. (Mirdh et a / . 1987). T h e parietal cells suspended in modified HBSS with 0.5 mM Ca2+ and O . I " / ~ BSA ["C1Aminopyrine (specific activity 102 mCi mrnol-.'), were elutriated through a Beckman JE-6B elutriator monoiodinated ['251]tyr1-somatostatin tetradecapepusing a rotor speed of 2000 r.p.m. The parietal cells tide ([""I]somatostatin) and ["51]Bolton-Hunterlabelled cholecystokinin octapeptide ([ 1251]BH-CCK- were recovered at a pump (Cole-Parmer Masterflex, 8) with specific activities of 2200 Ci mmol-' were Cole-Parmer Instr. Co., Chicago, IL, USA) flow of obtained from New England Nuclear (Dreieich, 55 ml min-'. T h e cells were subsequently collected by FRG). Percoll was bought from Pharmacia Fine centrifugation at 50 g for 5 min. Normally, 5-20 x 1o6 Chemicals, Uppsala, and Fluoroinert FC-77 from 3M cells were obtained, of which 30-507b were parietal Co., St Paul, IMN, USA, while Hanks' balanced salt cells. A Percoll density gradient was made by mixing 70 solution (HBSS) was from Gibco Biocult Ltd, Paisley, 6 in modified Hanks' BSS, giving a Scotland. HBSS was buffered at p H 7.4 with 2 5 rnM and 3 1 ~ Percoll Hepes. Other chemicals were of analytical grade. linear gradient with a density from 1.05 to 1.08 g mlV1. Bombesin, CCK-8 and somatostatin-14 were from Cells in 1-40 ml of modified HBSS with no calcium Peninsula Lab., San Carlos, CA, USA. Gastric were layered on top of the gradient and centrifuged at 1500g for 1 5 min. Fractions were obtained by inhibitory polypeptide (GIP) was a gift from Professor pumping Fluoroinert to the bottom of the tube and V. A4utt, Department of Biochemistry, Karolinska Institute, Stockholm, Sweden. collecting the fractions with lower densities. The Isolation of murosai cells. Porcine gastric cells were parietal cells were obtained by centrifugation at 50 g prepared as earlier described (hldrdh rt al. 1984, for 5 min. T h e yield was 1 - 1 0 x 106 cells, of which 1987). Stomachs were opened and rinsed and an up to around 800,; were parietal cells. approximately 60-cm2 large segment of mucosa from .4minopyrine uptake. Cells were incubated at 37 "C the corpus-fundus region was separated from the in HBSS with 10 mM glucose, 2 p.m ['4C]aminopyrine, muscularis layer and transferred to 500 ml of oxyge- 0.5 mM or 1 . 2 5 mM Ca2+ and various stimulants or nated Hanks' BSS without calcium. At the laboratory, inhibitors of acid secretion. At specified times, IOO pl the mucosa was scraped free from mucus and cut by of cell suspension was withdrawn and layered over a pair of scissors in I-cm-wide strips from which the 300 pl of ice-cold HBSS with 0.1yoBSA in microfuge submucosa was dissected away-. The mucosal strips tubes and centrifuged at 9000 g in a Beckman type B were then cut into small pieces which were placed in microcentrifuge for 1 5 s. T h e pellet was washed four j o ml modified HBSS without calcium but containing times and broken up in IOO pl roo; perchloric acid. 1 . 5 mg ml-' pronase in a Nalgene Erlenmeyer flask T h e mixture was centrifuged at 9000 g for 45 s and which was placed in a water bath at 37 "C for 10 min. the supernatant was dispersed in a liquid scintillation The material was stirred by a magnetic stirrer. solution consisting of I j parts toluene, five parts After the incubation, the undigested material was Triton X-IOOand one part Permafluor (New England allowed to settle and the supernatant was aspirated. Nuclear). Liquid scintillation counting was performed

Receptors on gastric mucosal cells in a Packard model Tricarb 2000 CA counter (United Technologies-Packard, Downer's Grove, IL, USA). One hundred-microlitre samples of the incubation suspension were also counted in order to determine total radioactivity. In a second series of experiments, after 45 min of incubation zoo-p1 samples were layered on 75 pl of silicone oil (density 1.03 g ml-') in microfuge tubes and centrifuged for 15 s (Mirdh et al. 1987). The tips of the tubes were cut off and placed in the liquid scintillation solution and counted. In this series of experiments, quench correction was carried out using an external standard. Binding of radioiodinated hormones. Cells in oxygenated HBSS with 1.25 mM Ca2+, 1.5% BSA, 0.5 mg ml-' bacitracin, z x I O - ~M captopril, 10-' M pepstatin A, I O - ~M phenylmethylsulphonylfluoride, 10-' M phosphoramidon and 0.3 mg ml-l of soy-bean trypsin inhibitor were incubated at various temperatures with approximately 3 x 10-'' M of [12'I]BHCCK-8 or ['251]somatostatin with or without unlabelled hormone. At specified times, duplicate 100-pl samples of the cell suspension were withdrawn and layered over 300pl of ice-cold buffer in microfuge tubes and spun for 15 s at gooog. The pellets were washed four times and then counted for radioactivity in a Packard 800 C gamma-counter together with 100-pl samples of the incubation suspension, counted for determination of total radioactivity. Tracer binding in the presence of 10-' M of unlabelled peptide is referred to as unspecific binding. Statistical analysis of data. Mean values for ['4C]aminopyrine uptake into parietal cells during stimulation with histamine alone (5 x IO-' M) or in combination with varying concentrations of somatostatin were compared with the Student's t-test for paired values. Comparisons of maximal ["C]aminopyrine uptake (as fraction of basal) into the initial preparation of mucosal cells and into elutriated cells were made with the t-test for unpaired values.

RESULTS Histamine time-dependently stimulated gastric mucosal cells to increase their accumulation of ['4C]aminopyrine (Fig. I). After 60 min, the uptake was up to 20 times above control levels in the presence of I O - ~M of histamine. There was no statistically significant difference (P x 0.5) between histamine-evoked relative increase of aminopyrine uptake into the initial preparation of mucosal cells and into elutriated cells. Mean uptake in response to maximal doses of histamine was 4.3& 1.6 pmol per 104 parietal cells of histamine. In a second series of [14C]aminopyrine ac-

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Fig. I. Time course for ['4C]aminopyrine uptake in 5 x 10' ml-' porcine parietal cells at 37 "C during stimulation with histamine control conditions (A-A), 10-' M (V-0) or I O - ~M (0-O), and inhibition by NaSCN z x I O - ~M (0-0). The data show results from one experiment representative of four similar experiments.

cumulation experiments, uptake in response to graded doses of CCK-8 and histamine were determined (Fig. 2). Histamine increased uptake to an average maximum of 445 Yoof control level at 5 x IO-* M histamine. The maximal response to CCK-8 obtained at 3 x IO-' M was 215% of uptake at basal conditions. Threshold concentrations of histamine and CCK-8 were 5 x IO-@ M and 10-l' M respectively (Fig. 2). Against a submaximal background stimulation of 5 x 10-' M of histamine, 3 x IO-' M of somatostatin caused a statistically significant 24 yo reduction of [14C]aminopyrine uptake. This inhibition was only slightly increased a t higher concentrations of somatostatin (Fig. 2). Somatostatin did not influence aminopyrine uptake at basal conditions (not shown). A fraction of elutriated gastric cells containing about 70 yo non-parietal cells bound specifically approximately 2 yo of added ['251]BH-CCK-8 (Fig. 3A), while a fraction containing around 60% parietal cells specifically bound 9% of

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available [lZ51]BH-CCK-8 (Fig. 3 B), corresponding to 0.4 and I .2% specific binding per 10' 5001 I cells respectively. About 50 yo inhibition of ["'I]BH-CCK-8 binding was obtained with 10-9M unlabelled CCK-8 (Fig. 4). Bombesin ( I O - ~M) did not reduce binding of CCK-8 (Table I ) while I O - ~M G I P and I O - ~M dibutyryl cyclic G M P inhibited binding to 22 yoand 60 yo of control levels respectively (Table I ) . ,\ preparation of elutriated cells containing 30 O0 parietal cells specificially bound up to 13 yo of added ['251]somatostatin (Fig. 5). Specific binding of ['251]somatostatin was also obtained with a preparation containing about 80 yoparietal cells. Again, lo-' M of unlabelled somatostatin caused approximately 50 yoinhibition of binding (Fig. 6). CCK-8 ( I O - ~M) did not affect binding of somatostatin (Fig. 6). In separate experiments, dissociation of bound [lZ5I]somatostatin was studied after dilution of 10 9 8 7 6 5 4 3 2 tracer with or without addition of an excess -Ig [ A g e n t ] , moll1 concentration of unlabelled somatostatin (Fig. Fig. 2. Effects of graded concentrations of CCK-8 7). T h e rate of dissociation of bound radioactivity (O--O),histamine (n-n) and somatostatin (0-0) per min was 1 . 3 % during an initial 30-min on [ "Claminopyrine uptake during .+jmin incubaperiod after dilution. With addition of an excess tions at 37 "C. The effects of somatostatin were of unlabelled peptide the dissociation rate studied against a background stimulation of aminop>rine uptake by j x I O - ~M histamine. The results are increased only slightly to 1.6% per min. presented as percentage of control uptake without added secretagogues or inhibitors. T h e data represent means of three separate experiments, each performed in triplicate. Vertical bars denote SE. Aminopyrine uptake influenced by histamine alone ( jx I O - ~M) or in combination with various concentrations of somatostatin was compared by t-test: * P < 0.05,

+*P < 0.01.

DISCUSSION The present study confirms earlier studies showing that isolated porcine gastric mucosal cells respond with a marked increase in aminoB

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Fig. 3. Time course for binding of [12'I]BH-CCK-8 to a preparation containing approximately 7 o 0 0 non-parietal (A) or 60°b parietal cells (B). Around 3 x 10-l' M of [lZ5I]BH-CCK-8 were incubated with j-8 x xo6 cells per ml a t 37 "C with (V-V) or without (0-0) I O - ~ M unlabelled CCK-8. 0-0denotes specific binding. T h e data are from one experiment.

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Fig. 5 . Time course for binding of ['251]somatostatin (3 x 10-'l M) to 10x 10' mucosal cells per ml, of which 30% were parietal cells. Cells were incubated at 22 "C. The curves represent total (0-O), unspecific (in the presence of IO-' M unlabelled peptide, &A) and specific (0-0) binding in one experiment of three similar experiments.

-10 [CCK-81, moll1

Fig. 4. Inhibition of binding of ['251]BH-CCK-8 by various concentrations of unlabelled CCK-8.3-6 x 10' mucosal cells per ml containing 30-60% parietal cells were incubated for 45-60 min at 37 "C with approximately 3 x 10-'' M ['251]BH-CCK-8. Data are means of two separate experiments. Vertical bars denote range.

Table I. Inhibition of binding of ['251]BH-CCK-8 by various peptides Unlabelled peptide

(M)

Binding of added radioactivity (yo)

0

-

1.20

CCK-8 CCK-8 CCK-8 GIP Bombesin dbcGMP

10-l'

1.15

10-*

0.28

10-'

0.13

10-'

0.26

10-'

1.19

IO-~

0.72

3 x 10-' cells per ml, of which 40% were parietal cells, were incubated at 37 "C for 60 min with approximately 3 x 10-'l M of [lZ5I]BH-CCK-8 and with various unlabelled peptides at specified concentrations.

11

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Fig. 6. Inhibition of binding of [1251]somatostatinby various concentrations of unlabelled somatostatin. 2 x 10' cells per ml, of which around 2 0 % were parietal cells, were incubated for 75 min at 22 "C with approximately 3 x 10-l' M ['2SI]somatostatin. Addition of 10-' M CCK-8 (0). The data are from one experiment. pyrine accumulation when stimulated with histamine (MHrdh et al. 1984, 1987). Furthermore, it is demonstrated that CCK-8 induces a similar but weaker response, while somatostatin moderately, but significantly, in-

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agrees fairly well with our present findings with ['"IIBH-CCK-8 as tracer and porcine cells as receptor preparation. In the canine parietal cells, CCK-8 was also found to be equipotent with gastrin-I 7 in stimulating aminopyrine accumulation (Soll et a / . 1984). Since the original submission of the present paper to the journal, Magous et al. (1989) have reported that gastrin17 and CCK-8 bind with similar affinity to a preparation of rabbit gastric cells containing 7500 parietal cells, whereas CCK-39 displays a lower affinity. 40t In the present study, there was a slight L discrepancy between concentrations of CCK-8 4 I reducing tracer binding (10-9M) and stimula20 40 80 ao 100 ting aminopyrine uptake (3 x 10-l") to 50% of Time (min) maximum, suggesting that not all CCK receptors Fig. 7. Dissociation of ['"jI]somatostatin from gastric need to be occupied to elicit maximal stimulation mucosal cells. z x 1 0 6 cells per ml (4010 parietal cells) of uptake. were incubated with approximately 3 x 10-l' b~ The effect of dbcGMP on CCK binding was ["51]sornatostatin at zz "C for 50 min. Cells were then studied, since it is known that dbcGMP specificentrifuged for 2 min at I 500 g after which cells were cally inhibits binding of CCK to its receptors in resuspended in buffer with (0-0) or without (0-0) unlabelled somatostatin ( I O - ~51). Binding is expressed the exocrine pancreas (Jensen et al. 1980, Sjodin as per cent of binding immediately after centrifugation. 1985). The present results show that dbcGMP also inhibits binding of CCK to sites on gastric The data is from one of two similar experiments. cells, supporting the notion that the observed sites are specific CCK receptors. Preparations of hibits the response to histamine but has little extracted porcine G I P have been reported to effect on basal activity. exert both secretin- and CCK-like effects on Preparations of porcine gastric mucosal cells isolated pancreatic acinar cells (Sjodin & Conlon have also been found to respond to gastrin with 1984). Since, in that study, synthetic G I P only increased aminopyrine uptake (Norberg et a / . evoked secretin-like effects, the CCK-like action 1986) and elevations of cytosolic Ca'+, levels on was probably due to a I "/b contamination of the addition of gastrin were recently detected in G I P preparation with CCK-33/39 (Sjodin & suspensions containing 85 O 0 parietal cells (Ca- Conlon 1984). Such a contamination is also a hero et ul. 1989). likely explanation for the presently observed The current results also show that porcine inhibition of CCK binding with a high congastric mucosal cells specificallj- bind cholecysto- centration of a porcine G I P preparation. kinin. A preparation of elutriated cells consisting Bombesin has been shown to evoke acid of around 600.~ parietal cells bound about three secretion in zivo (Erspamer & Melchiorri 1975). times more CCK per I O ~cells than a fraction This has been considered to be an indirect effect with 70 00 non-parietal cells, suggesting that the of bombesin mediated by release of gastrin observed binding sites are mainly located on the (Erspamer & Melchiorri 1975). However others parietal cells and not on other cells. However, have reported direct stimulatory effects of the possibility that some binding of ['"IIBHbombesin on acid secretion from isolated gastric CCK-8 on small endocrine cells occurred, as has mucosa (Ayalon et al. 1981). been suggested by others (Soll et a / . 1984, 1985), The lack of interaction of bombesin with cannot be excluded. These workers (Soll et al. CCK receptors in the present preparation 1984) also reported that CCK-8 competed with provides a further indication of the specificity of gastrin with equal affinity for canine parietal cell the studied binding sites. receptors. Fifty per cent inhibition of specific Specific somatostatin binding occurred to a binding of the gastrin tracer was obtained with purified preparation containing about 80 Yo approximately 6 x 10-'" .M of CCK-8, which parietal cells, making it reasonable to assume 1

Receptors on gastric niucosal cells

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that these* cells were an important source of binding, since such binding has been reported to somatostatin receptors. This does not exclude be an obstacle to delineating specific binding of the possibility that some of the observed binding, somatostatin to canine parietal cells (Park et al. particularly to less purified preparations, may 1987). Thus, fractions of isolated porcine mucosal have been related to other cellular elements of cells with a high proportion of parietal cells bind the gastric mucosa. Somatostatin receptors on parietal cells from specifically and with high affinity [12,1]BHrats (Reyl et al. 1979) and dogs (Park et al. 1987) CCK-8 and ['251]somatostatin, suggesting the have been reported. I n the recent study, Park existence of receptors for cholecystokinin and et al. obtained 50% inhibition of specific somatostatin on porcine parietal cells. Since both ["51](Le~s-~-Trp22-Tyr25) somatostatin-28 bind- peptides affect aminopyrine uptake into parietal ing with 6 x 10-' M of somatostatin-14, which is cells, it appears likely that the receptors are of a somewhat higher concentration than what was functional significance. The presently demonstrated stimulation of apparently needed for similar inhibition in the current study. However, Park et al. (1987) also aminopyrine uptake by CCK may be in agreedemonstrated that somatostatin inhibited his- ment with earlier in-vivo findings (Sjodin 1972b, tamine- and gastrin-stimulated ['*C]aminopyrine c) showing that CCK and caerulein are powerful accumulation in their preparation of enriched potentiators of vagally induced secretion in the parietal cells with an apparent IC,, of approxi- dog, elicited by sham feeding. It has been mately 4 x IO-' M. Similar findings have earlier reported that CCK releases somatostatin from been reported by Chew (1983) using a prep- preparations of gastric mucosal cells (Soll et al. aration of isolated rabbit gastric glands. I n the 1985) and that somatostatin release is inhibited present study, the inhibition of histamine- by vagal activation (Uvnas-Wallensten et al. stimulated aminopyrine uptake by somatostatin 1980, McIntosh et al. 1981). These findings may was of a similar magnitude as that reported by explain why CCK in vivo is a potent enterogastrone (Johnson & Grossman 1970) in the Park et al. (1987) in canine cells. The present experiments also demonstrate vagally denervated gastric mucosa, in which that binding of [1251]somatostatin to porcine somatostatin release may be uninhibited, and a gastric mucosal cells is, in accordance with powerful enhancer of vagally activated gastric previous findings (Park et al. I&), a reversible acid secretion (Sjodin 1972b, c), during which process which is not markedly accelerated by somatostatin levels are suppressed. addition of an excess of unlabelled hormone, The excellent technical and secretarial assistance by suggesting absence of negative co-operativity Hans Dahlkn, Tomas Bjorkman, Elisabeth Lindberg between binding sites (Kahn 1975). and Christina Karlsson is gratefully acknowledged. In earlier binding studies with canine cells This project was supported by the Swedish Medical iodinated gastrin-17 has been replaced by Research Council (grant no 4x-4965). ['251]Leu'5-gastrin-~7 (Soll et al. 1984) and REFERENCES somatostatin-14 by [1251]Le~8-~-Trp22-Tyr25somatostatin-a8 (Park et al. 1987) as tracers to AYALON, R., DEVITT, P.G., RAYFORD, P.L. A., YAZIGI, obtain or improve significant specific binding. & THOMPSON, J.C. 1981.Direct effect of bombesin The Leu" substitution in gastrin may have been on isolated gastric mucosa. Biochem Biophys Res of particular importance for avoiding inactivation Commun 99, 139-1397. BATZRI, S. & DYER,J. 1981. Aminopyrine uptake by of the gastrin molecule by oxidation of the Met" guinea pig gastric mucosal cells. Biochim Biophjis residue during oxidative iodination with the Acta 675, 416-426. chloramine T method (Hunter & Greenwood BERGQVIST, E. & OBRINK, K.J. 1979. Gastrin-hista1962). The present use of a CCK tracer labelled mine as a normal sequence in gastric acid stimuwith the Bolton-Hunter reagent reduces this lation in the rabbit. Up 3 Med Sci 84, 145-154. risk. The addition of a battery of enzyme BLOOM,S.R., MORTIMER, C.H., THORNER, M.O., inhibitors, such as bacitracin, phenylmethylsulBESSER,G.M., HALL,R., GOMEZ-PAN, A,, ROY, phonylfluoride, phosphoramidon and soy-bean V.M., RUSSEL, R.C.G., COY,D.H., KASTIN, A.J. & trypsin inhibitor, to the incubation medium in SCHALLEY, A.V. 1974. Inhibition of gastrin and the present binding studies was made in order to gastric acid secretion by growth hormone release inhibiting hormone. Lancet 2, 1106-1 109. minimize hormone degradation and unspecific

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