Suppression of somatostatin levels in the hepatic portal and systemic plasma of the rat by synthetic human pancreatic polypeptide

BIOCHEMICAL

Vol. 89, No. 3, 1979 August

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 913-918

13, 1’279

SUPPRESSION OF SOMATOSTATIN LEVELS IN THE HEPATIC PORTAL AND SYSTEMIC PLASMA OF THE RAT BY SYNTHETIC HUMAN PANCREATIC POLYPEPTIDE A. Arimura,

C.A.

Meyers,

W.L.

Case,

W.A. Murphy,

Tulane Departments of Medicine and Anatomy, and Endocrine and Polypeptide Laboratory, Hospital, New Orleans,

Received

June

and A.V.

University Veterans LA 70112

Schally

School of Medicine, Administration

29,1979 SUMMARY

Intravenous administration of synthetic human pancreatic polypeptide (hPP) with 36 amino acid residues significantly decreased immunoreactive somatostatin levels in both hepatic portal and systemic plasma in anesthetized rats. The effect of hPP on plasma somatostatin levels is acute. The results suggest thathPP may have a role in controlling somatostatin secretion from the gut and pancreas. Homologous hormonal Kimmel

pancreatic

properties et.al.

physiological

were

antisera

stimuli

which

amino

been

(RIA)

Ingestion

acids

increase suppresses

relationships

between

actively

studied,

has not

been reported.

by lack

of ample

pure our

hPP1--36 laboratory

(8,9,5).

plasma

the

which

effect

These

fat,

of plasma

(10).

On the

rise

and other

pancreatic

and related of pure

enabled

in

PP. high

secretion

studies

and for

of

hand,

of

and gut

infusion Although hormones

of somatostatin

have been the

yield

by a solid its

with

administration

hPP (10).

Recently,

us to investigate

secretion

hPP increase

other

of plasma

the

and hPP (7).

or intravenous

of PP on the

achieved has

levels

postprandial

somatostatin

quantities

has been

studies

by

for

(PP)

immunocytochemical

meal,

pancreas

polypeptide aPP (6),

Basal

with

The search

against

PP levels

the

residues)

and mammalian

pancreatic

for

acid

(4,5).

generated

of protein

somatostatin

are

used

avian

et.al.

affect

by antisera

have

(36 amino

from

and by Chance

radioimmunoassay age (10).

isolated

(1,2,3)

has been facilitated These

polypeptides

total

largely

hampered

synthesis phase

physiological

of

method

in

function.

Abbreviations: PP,pancreatic polypeptide; hPP, human pancreatic polypeptide; aPP, avian pancreatic polypeptide; RIA, radioimmunoassay; B.W., body weight; KIU, Kallikrein Inactivator Units;iv, intravenous; ip, intraperitoneal. 0006-291X/79/150913-06$01.00/0 913

Copyright All rights

@I (979 by Academic Press. Inc. ofreproduction in any form reserved.

Vol.

89,

No.

3, 1979

The structure

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

of hPP is as follows:

Ala-Pro-Leu-Glu-Pro-Val-Tyr-Pro-Gly-Asp-Asn-Ala-Thr-Pro-Glu-GlnMet-Ala-Gln-Tyr-Ala-Ala-Asp-Leu-Arg-Arg-Tyr-Ile-Asn-Met-Leu-ThrArg-Pro-Arg-Tyr-NH2 Furthermore, for

rat

we have

plasma

that

hPP,

from

the

somatostatin

unlike gut

recently

other

(11).

gut

the

overcome

This

hormones,

difficulty

paper

of radioimmunoassay

reports

suppresses

the

secretion

first of

observation somatostatin

and pancreas.

METHODS Young adult male rats of CD strain from Charles River Co., weighing They were housed in animal 240-300 g, were used throughout the experiments. quarters equipped with controlled light (on at 0500, off at 1900) and The animals were fed Purina rat diet ad libitum temperature (24 ?r 2OC). (Exp.1) or fasted overnight before the experiment (Exp.2). All rats were allowed access to water ad libitum. Rats were anesthetized with sodium pentobarbital (Nembutal), 5mg/lOOg body weight ip. Fifteen min after Nembutal, 0.5 ml 0.9% saline or hPP dissolved in saline was injected into the jugular vein over a period of 0.5 - 1 min. In Experiment 2, hPP was given iv as a bolus followed by an infusion over a 5 min period. Two or 5 min after the initiation of injection, 2 ml of blood was withdrawn from the jugular vein. The animal was then laparotomized and 2 ml of blood was collected from the hepatic portal vein. Blood collection from the jugular and the hepatic vein was completed within 2 minutes. Blood was placed in a chilled test tube containing 5 mg EDTA and 1000 KIU Trasylol (FBA Pharmaceuticals, New Yorkj. Plasma was separated by centrifugation at 2000 rpm at 4OC. To 0.5 ml plasma was added 0.5 ml 2M acetic acid, and then 4 ml cold acetone was added drop-wise to the acidified plasma while the test tube was being vortexed. The mixture was sonicated for 30 set and centrifuged at 3000 rpm for 10 min at 4OC. The supernatant was decanted into a 16 X 100 mm gelatin-coated tube. The precipitate was washed with 1 ml 80% acetone and the tube centrifuged. This supernatant was added to the gelatin-coated tube and washed with 3 ml of an organic solvent (ethyl acetate: ether = 3:l). After a brief centrifugation at 2000 rpm, the upper organic layer was aspirated and the aqueous acetone layer was dried by nitrogen gas at 37-40°C. The dried residue was dissolved in 0.5 ml diluent for RIA, and 0.2 ml was assayed for somatostatin by RIA using rabbit antiserum to somatostatin (RlOl) as described elsewhere (11). hPP was synthesized by a stepwise solid phase method with modifications similar to those recently described for the synthesis of somatostatin and other peptides (12-14). Details of the synthesis and purification of hPP will be reported elsewhere. Mean plasma somatostatin levels of each treatment and compared to each other using Duncan's new multiple

group range

were test

has been

found

calculated (15).

RESULTS AND DISCUSSION The extraction almost teins

all

interfering

and degrading

method

for

plasma

substances enzymes.

This

somatostatin

in the method

914

rat

plasma

can be also

including applied

to eliminate

binding

pro-

to measurement

BIOCHEMICAL

Vol. 89, No. 3, 1979

TABLE

1.

Effect of somatostatin

Substance injected

(pg/ml) Portal

plasma

(5) (5)

72.0 27.8

f f

Exp.

2.

Saline hPP

5.4 2.9

+ 1.8 ‘I 0.38

(5) (4)

73.3 29.5

t 14.1d + 2.10e

In

Exp.l.,

10 pg hPP/lOO g B.W. collected 2 min after

In

Exp.Z.,

5 pg hPP/lOO iv infusion

of

was injected the initiation

g B.W. was 5 ng/lOO

iv

over 1 min. of injection.

injected iv as a bolus g for a 5 min period.

10.81s 9.08c

Blood

(5) (5)

(4) (4)

was

followed

by

vs systemic plasma saline group, PcO.01; b: vs corresponding saline group, PcO.05; c: vs corresponding saline group, PcO.02; vs corresponding systemic plasma group, P< 0.05; vs corresponding systemic blood, PcO.01; e: vs corresponding systemic blood, P~0.01; vs corresponding saline group, P~0.01.

of

added

ml

averaged

for

the

in

parenthesis

in

Mean

The

limit

of

or

2.5-5

control

plasma

were

in

hepatic values

systemic source

and portal

were

of

95%

confidence

limits

5.4

plasma levels

pg/ml

plasma

in were

plasma,

indicating

circulating

reduction

200

in

acetone

Exp.

1 and

72.0

and

of

somatostatin

~1

times

that

the

gut

915

plasma

Exp.

hepatic

2,

recovery

and

500

pg

by

the

amount is

systemic

respectively.

Those

respectively; than

the

per

used. of

pg/ml,

and

The

control is

greater

1J-g synthetic in

buffer

extracts

(Table 10

100,

determined

the

73.3

14

(16). 20,

as of

7 to

of

of

RIA

when

somatostatin

administration

modification doses

sensitivity

pg/ml

rats.

a slight in

approximately

Intravenous 60%

with

of

assay

somatostatin

10.4

of

blood

number

tetradecapeptide

99%. lower

indicate

whole

somatostatin

pgltube,

imately

levels 2 SE plasma

+ 1.36 + 0.93b

somatostatin

major

Somatostatin mean Systemic

hPP on plasma

10.4 6.6

of

the

synthetic

Saline hPP

Numbers

these

of rats.

in

1.

d:

the

administration levels

RESEARCH COMMUNICATIONS

Exp.

a:

0.5-l

AND BIOPHYSICAL

the

pancreas

levels are

in the

1). hPP portal

resulted plasma

in and

approxabout

40%

Vol.

89,

No.

3,

reduction

1979

in the

in Experiment only not

BIOCHEMICAL

about

systemic

2 where half

systemic

2 min changes the

portal

plasma

plasma

levels

1,

reduction

the (Exp.

1).

2) both

However,

of somatostatin

were

in somatostatin

1) and a bolus induced

was

injection

similar

followed

suppressive

in the

somatostatin

administration

levels

rat.

levels

the

in normal

the of

approximately moment to moment Therefore,

animals.

by hPP observed

prompt

hPP

half-life

short,

may reflect

somatostatin

may indicate

in both

Since

is extremely

the hormone

of plasma

10 ug synthetic

somatostatin

in vivo -___

ratesof

reduction

following

plasma

iv-administered

reduced

in secretory

acute

show that

immunoreactivity the

(Table

one min

residues

and hepatic

(17),

experiments

of hPP (Exp.

clearly acid

somatostatin

in both

COMMUNICATIONS

somatostatin.

The results 36 amino

RESEARCH

systemic

over

infusion

on plasma

BIOPHYSICAL

in Experiment

Infusion

by a five-minute

with

control

of those

significant.

effects

plasma

AND

2-5 min

suppression

of

secretion

of

somatostatin. Various

gut

hormones

somatostatin

(18).

suppresses

somatostatin

pharmacological.

hPP is

protein

meals

or in the several attain

less

times

considerably 10 pg hPP/lOO

hormone The dose

the

stimulate

so far used

suppressive

of hPP in systemic 100 pg/ml

and fat

(10).

circulation greater

such high

Although

release.

than

local

only

glucagon

reported

in this

release

of

which

clearly

study

action

may not

in young

people

may be necessarily

significance.

levels

by RIA are

the

Therefore,

be of physiological Basal

and pancreatic

large

hPP on gastrointestinal

hPP is

studies

may not (10,

functions,

above in the

secreted

concentration

clear-cut

suggest

500 pg/ml

likely

portal

after blood

to be at least

systemic

to be given.

19-21)

as determined

hepetic

by systemic

be excessive

916

is

in the

tissue

of hPP may have

g body weight several

the

in a local

dose

and increase

The concentration where

than

levels

(lo),

plasma

blood.

administration,

TO a

Accordingly,

in this possible

physiological

regard. influence actions

of of PP

Vol.

89,

still

No.

3, 1979

remain

release of

elusive.

of various

antiserum

gesting

PP-cells

other

but

tumor,

release

high rose This

COMMUNICATIONS

known to suppress Administration

PP. PP levels

(22),

by somatostatin

takes

to somatostatin

in the

sug-

producing

peripheral

part

of

throughout

the

exocrine

parenchyma

control

the

secretory

activity

and F-cells

(23).

of

fashion.

we observed

extremely

scattered

D-cells

plasma

Similar

localized

is

including

of PP secretion

are

RESEARCH

somatostatin

increased

conditions.

in a paracrine

detectable, of the

control

BIOPHYSICAL

hormones,

in dogs

and also that

Recently showed

and pancreatic

(F-cells)

islets

is possible

each

hand,

physiological

pancreatic It

On the other

inhibitory

under

D-cells,

AND

to somatostatin

that

place

gut

BIOCHEMICAL

that

in a patient

circulating to Spg/ml, finding

PP levels, which

supports

by hPP has physiological

with

is the

a PP-cell

plasma

that

significance

who

somatostatin

in a low normal view

tumor

was un-

range,

after

suppression

of

(unpublished

study

removal

somatostatin with

Dr.

R. Friesen).

ACKNOWLEDGEMENTS 22156

This study was supported (CAM), and the Veterans

in part by NIH grants Administration.

AM 09094

(AA)

and AM

REFERENCES 1. Kimmel, 83,

J.R.,

Pollock,

H.G.,

and Hazelwood,

R.L.

(1968)

Endocrinology

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2. Kimmel, J.R., Pollock, H.G., and Hazelwood, R.L. (1973) In "Atlas of Protein Sequence and Structure" (M.D. Dayhoff,Ed.) Vol. 5, Suppl. 1, p. 5, Natl. Biomed. Res. Found., Washington, D.C. 3. Kimmel, J.R., Hayden, L.J., and Pollock, H.G. (1975), J. Biol. Chem. 250,

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4. Chance, R.E. (1972) Diabetes, 21, Suppl. 2, 536. 5. Chance, R.E., Lin, T.M., Johnson, M-G., Moon, N.E., Evans, D.C., Jones, W.E., and Koffenberger, J.E. (1975) 57th Annual Meet. Endocr. Sot. Abstract No. 265, p. 183. 6. Langshow, D.R., Kimmel, J.R., and Pollock, H.G. (1973) Endocrinology 93,

558-565.

7. Chance, R.E., Moon, N.E., and Johnson, M.G. (1979) In "Methods of Hormone Radioimmunoassay" (B.M. Jaffe and H.R. Behrman,Ed.), pp. 657-672, Academic Press, New York. 8. Floyd, J.C., Jr., and Fajans, S.S. (1976) Diabetes, 25, Suppl. 1, 330.

9. Adrian, (1976:)

T.E., Bloom, Gut, 17, 393.

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Floyd, J.C., Jr., Fajans, S.S., Pek, S., and Chance, R.E. (1977) Rec. Progr. in Hormone Res. 33, 519-570 Arimura, A., Lundqvist, G., Rothman, J., Chang, R., FernandezDurango, R., Elde, R., Coy, D.H., Meyers, C., and Schally, A.V. (1978) Metabolism 27, 1139-1144. Meyers, C.A., Arimura, A., Cordin, A., Fernandez-Durango, R., Coy, D.H., Schally, A.V., Drouin, J., Ferland, L., Beaulieu, M., and Labrie, F. (1977) Biochem. Biophys. Res. Commun., 74, 630-636. Meyers, C.A., Coy, D.H., Huang, W.Y., Schally, A.V., and Redding, T.W. (1978) Biochemistry, 17, 2326-2331. Coy, D.H., Gill, P., Kastin, A.J., DuPont, A., Cusan, L., Labrie, F ., Britton, D., Fertel, R. (1977) Proc. 5th Amer. Peptide Symp. in Peptides,(M. Goonman and J. Meienhofer, Eds.)pp. 107-110, Halsted Press, New York. Steel, R.G.D., and Torrie, J.H. (1960) in "Principles and Procedures of Statistics" ,p. 107, McGraw-Hill, New York. Chihara, K., Arimura, A., Schally, A.V.(1979) Endocrinology 104, 1434-1441. Kastin, A.J., Coy, D.H., Jacquet, Y.,Schally, A.V., and Plotnikoff, N.P. (1978) Metabolism, Vol. 27, 9, Suppl. 1, 1247-1252. Unger, R.H., Dobbs, R.E., and Orci, L. (1978) Ann. Rev. Physiol. 40, 307-343. Lin, T.M. and Chance, R.E. (1978) in Gut Hormones, (S.R. Bloom, Ed.), pp. 242-246, Churchill Livingston, Edinburgh. Adrian, T.E., Bloom, S.R., Besterman, H.S., and Bryant, M.G. (1978) ibid., pp. 254-260. Adrian, P.E., Greenberg, G.R., Besterman, H.S., McCloy, R.F., Chadwick, V.S., Barnes, A.J., Mollinson, C.N., Baron, J.H., Abberti, K.G.M.M., and Bloom, S.R. (1978) ibid., pp. 265-267. Schusdziarra, V. , Rouiller, D., Arimura, A., Boden, G., Brown, J., Unger, R. (1979) Diabetes, Suppl.2, in press. Larsson, LL., Sundler, F., and Hakanson, R. (1966) Diabetologia 12, 211.

12. 13. 14.

15. 16. 17. 18. 19. 20. 21. 22. 23.

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