Vol. 82, No. 2, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

May 30,1978

Pages 731-737

SOMATOSTATIN RELEASE FROM ISOLATED PERFUSED RAT STOMACH T. Chiba, H. Abe,

Y. Seino,

Y. Kato,

Y. Goto,

S. Kadowaki,

S. Matsukura,

T. Taminato,

M. Nozawa* and H. Imura**

Third Division, Department of Medicine, Kobe University School of Medicine, Kusunoki-cho, Ikuta-ku, Kobe 650, * Department of Osaka Medical School, Daigaku-cho, Takatsuki 569 and Surgery, ** Second Division, Department of Medicine, Kyoto University School of Medicine, Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606, Japan Received

April

18,

1978 SUMMARY

Gastric somatostatin release from the isolated rat stomach was studied using a perfusion technique. Somatostatin released from the isolated perfused rat stomach was found to be identical in molecular size and inununoreactively with synthetic somatostatin. Infusion of glucagon (10D7M) caused biphasic increase of gastric somatostatin release. Gastric somatostatin release was also stimulated Fy infusion of theophylline (10m3M) and dibutyryl cyclic AMP (10 M). These results indicate the possible involvement of adenylate cyclase-cyclic AMP system in the regulatory mechanism of gastric somatostatin release.

INTRODUCTION Immunoreactive the central (3,

nervous

4) and the

enylate

system

stomach

cyclase-cyclic

somatostatin

(3).

(1,

the

The present inununoreactive the

release study

2),

AMP system

demonstrated but

also

not

a possible

role

in the release

(5,

6).

was performed

of adenylate

of

in

of ad-

pancreatic

little

However,

is

somatostatin.

to examine

from perfused

only

in the pancreas

mechanism of gastric

somatostatin involvement

is

Recently,

has been suggested

known about

elucidate

somatostatin

rat

the release

of

stomach and to

cyclase-cyclic

AMP

OOQ6-291X/78/0822-0731$01.00/0 731

Copyright 0 I978 by Academic Press, fnc. AlI rights of reproduction in any form reserved

Vol. 82, No. 2, 1978

system

BIOCHEMICAL

in the regulatory

AND BIOPHYSICAL

mechanism of gastric

RESEARCH COMMUNICATIONS

somatostatin

release. MATERIALS AND METHODS Stomachs were isolated from 24 h fasted Perfusion of stomach: male Wistar rats weighing 300-350 g by the metnod of Lefebvre Polyethylene cannulae were inserted into the left gastric (7). artery and the gastric vein. All other vessels were ligated and a whole pancreas was carefully excluded by ligation. After a catheter was inserted into the stomach cardia was ligated, throu h the pyloric ring to drain the gastric juice. 4.6 S DKRBG7 was infused into the left gastric artery at a rate of 2 ml/min without recirculation. The perfusate was equilibrated immediately before use with a gas phase of 95 % O2 / 5 % CO2 at 37OC and the stomach preparation was kept at 37OC throughout the experiment. After the pre-perfusion period (20 min) when 4.6 % DKRBG was infused, 4.6 % DKRBG with or without various agents was infused into the artery for 15 min. The venous effluents were collected every 1 min in chilled tubes containing Bacitracin-Trasylol mixture (2 x 10m5M and 1000 U/ml, respectively), frozen immediately and stored at -20°C until assayed. 20 ml of the perfusate was pooled and Gel chromatography: shaked gently for 1 min with 2 volumes of 98 % acetone containing 0.35 N acetic acid. After centrifugation at 3500 rpm for 30 min, the supernatant was evaporated under air to remove acetone and then lyophilized. The residue was reconstituted to 1.5 ml with 0.2 N acetic acid and applied to Sephadex G-25 column (1.8 x 70 cm). Each 2 ml fraction was collected using 0.2 N acetic acid as an elution buffer, and eluates were assayed for immunoreactive somatostatin. Radioimmunoassay: Somatostatin levels were measured by a specific radioimmunoassay with a modification of the method described by Arimura et al. (9). Specific antiserum (RA-623) to somatostatin was produced in the rabbit by repeated immunization with synthetic somatostatin (Protein Researc!l Institute, Osaka, Japan) coupled to crystallized bovine serum albumin (Armour Pharmaceutical Co. Chicago). Antiserum RA-823 was found to have no cross-reactivity with insulin, glucagon, secretin, gastrin, motilin, VIP, substance-P, bombesin, met5enkephalin, ACTH, TRH, LH-RH and TSH. N-tyrosyl somatostatin was iodinated with lz51-Na by chloramin-T method (8) subsequently purified on Sephadex G-25 column. The minimum detectable quantity of the assay was 10 pg/ml (Fig. 1). An intraassay and an interassay variation were 5.4 % and 8.5 %, respectively. 1Abbreviations: DKRBG, dextran Krebs-Ringer bicarbonate buffer (pH 7.4) containing 5.5 mM glucose; VIP, vasoactive intestinal polypeptide; TRH, thyrotropin releasing hormone; LH-RH, luteinizing hormone-releasing hormone; TSH, thyroid stimulating hormone; db-CAMP, N6, 02-Dibutyryladenosine-3',5'-cyclicphosphate, Sodium Salt.

732

Vol. 82, No. 2, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Irmnunoreactive insulin was measured by radioimmunoassay with a polyethylene glycol method described previously (10). Immunoreactive glucagon was determined by radioimmunoassay with a talc adsorption technique (11) using antiserum 30 K. N-tyrosyl somatostatin was kindly supplied from Drs. D.H. Coy and A. Arimura, V.A. Hospital, New Orleans. Antiserum 30 K was purchased from Daibetes Research Fund, Dallas. Theophylline and db-CAMP were purchased from Nakarai Chemicals, Ltd., Kyoto and Sigma Chemical Co., St. Louis, respectively. Secretin, motilin, VIP, substance-P, bombesin (generous gifts from Dr. N. Yanaihara, Shizuoka College of Pharmacy, Shizuoka), human gastrin I (a gift from I.C.I. Ltd. Macclesfield Cheshire), human ACTH (supplied from Takeda Chemical Industries Ltd., (purchased from Protein Research InOsaka), met5-enkephaline stitute, Osaka), TRII, LH-RH (obtained from Tanabe Pharmaceutical co., Osaka) and TSH (purchased from Armour Pharmaceutical Co., Chicago) were of synthetic origin. Highly purified pork insulin and crystalline beef-pork glucagon were supplied from NOVO, Copenhagen and Lilly Lab., Indianapolis, respectively. RESULTS Three from

peaks of

immunoreactive

the perfusate

of

Sephadex

G-25 column

the void

volume

tion

of

the stomach by gel chromatography (Fig.

of

somatostatin curve

basal

somatostatin

217 pg/ml,

which

were not

the infusion

Infusion

somatostatin pg/ml

of

release

(mean i- SE)

the

(3 min after

second of

758 ? 101 pg/ml

(Fig.

3),

both

basal

levels

of which (P < 0.01

to the

1). levels

ranged

significantly

of DKRBG (mean + SE, 183 ?r 2 pg/ml)

glucagon

with

of immunoreacparallel

study,

changed

3).

curve

(Fig.

to

second peak.

to be completely

assay system

from 160 pg/ml

near

at a posi-

by a small

dilution

on

peak was eluted

in the main peak was quite

In the perfusion

(Fig.

preceded

A serial

in the

during

The first

the main peak was found

to charcoal.

standard

2).

somatostatin

Immunoreactivity

tive

were obtained

and the main peak was demonstrated

synthetic

adsorbed

somatostatin

(10s7M) first the

peak value start

(15 min after

were significantly and P < 0.001,

733

evoked

of the

biphasic of

1024 + 152

infusion) start greater

respectively).

and the

of

infusion)

than

the

After

the

BIOCHEMICAL

Vol. 82, No. 2, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

B/F 09 Dilution 130

0.6

of extract 1:&o 1:20

of perfusate 1:10 1:s

76

31.2

07 0.6 0.5 0.4 0.3

‘:\

0.2 0.1 0

1

1.0

2D

3.9

156

Somatostatin

625

125

250

500

(pgltube)

Fig.

1:

Dose response curve of radioimmunoassay for somatostatin and dilution curve of the extract of perfusate Antiserum RA-823 was used from isolated rat stomach. Nonat a final concentration with 1:8000 dilution. specific binding of the assay after treatment with dextran-coated charcoal was 2.4 %. B and F show bound Standard somatoand free radioactivity, respectively. ) and the extract from gastric perfusate statin (oe (o----o) are shown.

Fig.

2:

Gel chromatography of the extract of perfusates from isolated rat stomach on Sephadex G-25 column (1.8 x 70 cm) in 0.2 N acetic acid at a flow rate of 25 ml/h. The column was calibrated with synthetic somatostatin (the cross-hatched bar) and dextran blue (void volume = Vo). Each fraction volume was 2 ml. 734

Vol. 82, No. 2, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Time(min)

Fig.

3:

end of

Effects of glucagon (10V7M), theophylline (10s3M) and db-CAMP (10s3M) on immunoreactive somatostatin release from isolated perfused rat stomach. All drugs were perfused for 15 min following a pre-perfusion period (20 min) as shown. Heavy solid lines (-) represent somatostatin responses to stimuli and light solid lines (0~ ) show the control study (glucose only). Mean rt SE values are shown. Number of animals is 6 for each group. Student's "t" test was used for statistical analysis and shown as follows : * P < 0.05, ** P < 0.005 compared with the control group.

infusion,

pre-perfusion

level

theophylline statin

somatostatin

(10 -3M)

release

after

the

with

start

db-CAMP (10 -3M) release

within

with

15 min after

of

6 min

produced

infusion

Immunoreactive

insulin

of

of

somatoat 11 min

(Fig.

3).

Infusion

rise

of

somatostatin

644 f 34 pg/ml

infusion

of

685 ?r 135 pg/ml

a gradual of

to the

Infusion

increase

(P < 0.01)

the peak value of

3).

a gradual

elicited

start

were decreased

(Fig.

the peak value

also

the

levels

(Fig.

and glucagon

(P < 0.001)

of

at

3). were not

detected

any perfusate. DISCUSSION The present statin

study

was released

revealed

from

that

isolated

immunoreactive

perfused

735

rat

somato-

stomach and

in

BIOCHEMICAL

Vol. 82, No. 2, 1978

responded

to various

ered

to be identical

with

synthetic

tive

somatostatin

synthetic serial

curve

studies

was parallel (5,

of pancreatic

somatostatin

release,

of

cyclase-cyclic

adenylate

anism of It

gastric could

since

pancreatic

fluenced study

suggests,

gastric not

detectable

especially release

however,

might

al.

be a regulator

somatostatin various

(13,

stimuli

somatostatin

it

release,

is

that

the

with

in

involvement mech-

(12).

to be inThe present

in

the case of were

stomach throughout gastric

the

hormones, somatostatin

future. pancreatic

homeostasis.

a possible

stomach,

and glucagon

gastric

that

from the

release

the

reported

other

in the

of nutrient

emphasizes

is

insulin

of

14) suggested

was released

showed

gastric

somatostatin

unlikely

since

may interact

in nutrient

study

the regulatory

the pancreas

that

be elucidated

cyclasemechanism

possible

in

gastric

release

The possibility

should

adenylate

The present

the

and its

curve.

regulatory

and glucagon

hormones in

gastrin,

Ipp et --

that

in the perfusates

experiments.

in the

to

release.

somatostatin

somatostatin

standard

that

also

by insulin

by these

to the

AMP system

be considered

be influenced

corresponding

and db-CAMP stimulated

indicating

immunoreac-

G-25 chromatography

release.

somatostatin

might

fractions

be involved

theophylline

glucagon,

and immunoreactively

6) demonstrated

somatostatin

was consid-

the main peak of

in the

on Sephadex

RESEARCH COMMUNICATIONS

somatostatin

size

since

was eluted

AMP system might

that

Gastric

in molecular

somatostatin

Recent cyclic

stimuli.

somatostatin,

dilution

AND BIOPHYSICAL

somatostatin

The fact

stomach responding involvement

that to

of gastric

homeostasis. ACKNOWLEDGEMENTS

We are grateful to Drs. D.H. Coy and A. Arimura, Hospital, New Orleans, for a g.enerous gift of N-tyrosyl

736

V.A.

Vol. 82, No. 2, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

somatostatin. We are also indebted to Prof. College of Pharmacy, Shizuoka, for synthetic hormones.

N. Yanaihara, a kind supply

Shizuoka of various

REFERENCES 1. Brazeau, P., Vale, W., Burgus, R., Ling, N., Butcher, M., Rivier, J. and Guillemin, R. (1973) Science 179, 77-79. 2. Hockfelt, T., Effendic! S., Johansson, O., Luft, R. and Arimura, A. (1974) Brain Res. 80, 165-169. 3. Arimura, A., Sato, H., Dupont,A., Nishi, N. and Schally, A.V. (1975) Science 189 1007-1009. -' 4. Dubois, M.P. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1340-1343. 5. Barden, N., Urbina, G-A., C6te, J.P. and DuPont, A. (1976) Biochem. Biophys. Res. Commun. 71, 840-844. 6. Schauder, P., McIntosh, C., Arends, J., Arnold, R., Frerichs, H. and Creutzfeld, W. (1977) Biochem. Biophys. Res. Commun. 2, 630-634. 7. Lefebvre, P.J. and Luyckx, A.S. (1977) J. Clin. Invest. 59 -' 716-722. 8. Hunter, W.M. and Greenwood, F.C. (1962) Nature 194, 495-496. 9. Arimura, A., Sato, H., Coy, D.H. and Schally, A.V. (1975) Proc. Sot. Exp. Biol. Med. 148, 784-789. 10. Goto, Y., Seino, Y., Tarnina% T. and Imura, H. (1976) J. Endocrinol. 69, 295-296. 11. Sakurai, H. and Imrua, H. (1976) Japan J. Nucl. Med. 10, 135-136. 12. Unger, R.H. and Orci, L. (1977) Diabetes -26, 241-244. 13. Ipp, E., Dobbs, R.E., Arimura, A., Vale, W., Harris, V. and Unger, R.H. (1977) J. Clin. Invest. 60, 760-765. 14. Ipp, E., Dobbs, R.E., Harris, V., Arimura, A., Vale, W. and Unger, R.H. (1977) J. Clin. Invest. 60, 1216-1219. -

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