Gastrointestinal somatostatin in man and dog

Gastrointestinal C. McIntosh,

Somatostatin

in Man and Dog

R. Arnold, E. Bathe. H. Becker, J. Kobberling,

and W. Creutzfeldt

T

HE CONCEPT of a role for somatostatin in gastrointestinal physiology has developed from several observations. Infusion of somatostatin is capable of suppressing pancreatic and gastrointestinal hormone release,‘,” gastric secretion,” pancreatic juice and enzyme secretion,4 and gallbladder contraction.’ Immunohistology has revealed a widespread distribution of somatostatin-containing cells in pancreas, stomach, duodenum, and jejunum”,” and the D cell appears to be the specific locus in pancreas and gastric antrum.“,” Radioimmunoassay has confirmed the gastrointestinal localization in rat7-“’ and chicken.” The demonstration of secretion of somatostatin from isolated rat pancreatic islets incubated in vitro”-‘3 implies a regulatory role for this polypeptide. The present report examines the distribution of immunoreactive somatostatin in the gastrointestinal system of dog and man and the question of multiple molecular forms in these organs. MATERIALS

AND METHODS

Assay Antibodies were raised in New Zealand white rabbits against synthetic cyclic somatostatin conjugated to bovine serum albumin by the carbodiimide technique.” The conjugate was emulsified in Freund’s complete adjuvant and injected intradermally. I-Tyr-somatostatin was iodinated using chloramine-T and the labeled peptide was separated from lrsI by absorption to QUSO G32 and elution with acetone/acetic acid. Prior to each assay, label was purified by CM-cellulose chromatography.’ Incubations were performed in 50 mM barbital buffer, pH 7.4 containing merthiolate (O.Ol%), aprotinin (500 KIU/ml), and I % human serum albumin. Following incubation for 48 hr at 4” C, separation of bound and free antigen was achieved with dextran-coated charcoal. Using antibody VI/3/i at a dilution of I: 12,500, levels of 300 fg per assay tube can be detected with 95% confidence. No cross-reaction occurs with other gastrointestinal hormones up to I pg. Intraassay variations are 7% and 4%. and interassay variations are 16% and l2%, respectively.

Extraction Human stomach antrum and corpus biopsy specimens were immediately frozen on dry ice. Resected pancreas was collected and frozen as soon as possible after operation. Biopsy specimens from dogs were collected under anesthesia. All specimens were stored at -20°C. Frozen tissue was quickly weighed, boiled for I5 min in 0.2 A4 acetic acid, and homogenized. Solid material was removed by centrifugation (10,000 g for IO min) and the clear supernatant lyophilized. For assay the extract was dissolved in distilled water and suitably diluted with buffer.

RESULTS AND

DISCUSSION

Somatostatin concentrations in gastrointestinal tissues from dog and man are given in Table I. In the dog, highest levels were found in the antrum and decreasing amounts as follows: pancreas (head), pancreas (processus uncinatus), pancreas (tail), stomach

From the Department of Medicine, Division of Gastroenterology, University of Gdttingen. Gb;ttingen, Federal Republic of Germany. Address reprint requests to Dr. C. McIntosh, Deparrment of Medicine, Division of Gasrroenterology, University of GGttingen. GiAtingen. Federal Republic qf Germany. :U 1978 by Grune & Stratton, Inc. 0026-0495/78/27/3-0035$01.00/0 Metabolism,

Vol. 27. No. 9.

Suppl. 1 (September).

1978

1317

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MCINTOSH

ET AL

Table 1. Somatostatin Concentration in Gastrointestinal Tissues from Man and DOP*

Stomach

antrum

493

Stomach.

corpus

195t33L6)

Pancreas,

head

333

*

66 (41

222-650

Pancreas

processus

205

*

41

(4)

122-310

Pancreas.

tall

187

i

27

(4)

138-265

98

*

17 16)

36-147

614

i

125

23-2250

478

i

170(16)

Duodenum.

uncinatus

proximal

*

197-900

114(6)

76-315

Human Stomach.

antrum

(duodenal

ulcer)

(24)

corpus

Stomach:

[duodenal

ulcer)

19~2500

Pancreas Hrstologically Chronic

normal

pancreatrtrs

Neonatal

253

+ 43

270

f

hypoglycemra

expressed normal Morrison

were

collected

as mean

pancreas syndrome

*

was

SEM obtained

31-640

(7)

221425

4.840

(Nesidioblastosrs) ‘Tissues

(15)

196

3.200 Adenomatosrs

and

extracted

and

range.

from

(1).or rnsulinoma

as descrrbed Numbers

accident

cases

in the

in brackets

Materials

indicate

(2) or patrents

wrth

the

and

5,500 Methods

number

Zollinger-Ellrson

section.

of extracts. syndrome

Levels

are

Hrstologrcally (1). Verner-

(11).

(corpus), proximal duodenum. Since levels of somatostatin in rat stomach antrum and corpus are approximately equalY (and unpublished observations), it appears that there are species differences in the relative distribution. With human material, a broad intersubject variation in both control and pathological samples is evident. Concentrations in human antrum from duodenal ulcer patients were similar to those found in dog but the levels in human stomach corpus were significantly higher and approached those found in antrum. It is obviously of interest as to whether tissue levels in duodenal ulcer patients differ from normal, since this could contribute to the etiology of the disease: however, data available at present do not substantiate this proposal. Normal human pancreas, despite the wide range, contained mean levels similar to those in dog. In the small group of pancreatitis patients, five had levels under 80 ngfg tissue and the other two patients 305 and I.425 ngfg tissue. Although pancreatic specimens were collected as soon as possible after operation, inevitable delays may partly contribute to the wide variation, since tissue somatostatin is degraded unless the specimen is rapidly cooled. Nevertheless, a possible relationship between degree of pancreatitis and tissue content is possible and is at present under investigation. The extremely high levels in the two children with nesidioblastosis (and surgicaily proven islet cell hyperplasia, including B, A, and D cells) may be characteristic for the pancreas in neonatal hypoglycemia. However, the nonavailability of normal tissue from babies makes this proposal difficult to verify. Column chromatography profiles of pancreatic extracts from rat, dog and man are shown in Fig. 1. A small peak eluting in, or just after, the void volume and a major peak eluting at the same position as synthetic tetradecapeptide somatostatin are present in all three species. With extracts from stomach antrum and corpus (not shown), similar profiles are obtained and, although the void volume

GASTROINTESTINAL

SOMATOSTATIN

Fig. 1. Gel chromatography of pancreatic extracts. Chromatography was performed on columns of Sephadex G-25 fine (100 x 1 cm). Elution was with 0.2 M acetic acid at a flow rate of 5 ml/hr. Collected fractions (1 ml) were lyophilized and dissolved in assay buffer. Void volume (Vo) and total volume (Vtl were determined using blue dextran and 12’1, respectively. SOM indicates the elution position of synthetic cyclic somatostatin.

peak is of variable size, no extract so far examined has contained more “big” somatostatin than “normal” somatostatin. Multiple species of somatostatin in brain”’ and gastrointestinal” tissue extracts have been previously reported. In particular, Arimura et al.” observed that rat pancreas contained the majority of somatostatin in a “big” form, which eluted in the void volume, while with stomach extracts the reverse occurred. Chromatography of pigeon pancreatic” and human somatostatinoma’,’ extracts revealed little immunoreactivity in the void volume. The latter, however, showed considerable heterogeneity and suggests the formation of other species by the tumor. Among the possible explanations for the different results are: The antibodies used by different workers are directed against different antigenic determinants, differences in the nutritional state of the animals and in extraction technique, or less nonspecific protein interference in particular assays. These questions remain to be resolved. The significance of gastrointestinal somatostatin and of the possible multimolecular forms is at present unknown. The fact that secretion occurs, at least from pancreatic islets, suggests that it is physiologically important, and investigations of tissue content and possible changes under different nutritional conditions or in pathological states may throw some light on its role in the gastrointestinal tract.

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