Somatostatin precursors: Evidence for presence in and release from rat median eminence and neurohypophysis

Vol. 90, No. 2, 1979 September

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

27, 1979

Pages 466-472

SOMATOSTATIN

PRECURSORS : EVIDENCE FOR PRESENCE IN AND

RELEASE FROM RAT MEDIAN EMINENCE AND NEUROHYPOPHYSIS Hans H. Zinggl

and Yogesh

C. Pate12

Fraser Laboratories, McGill University Departments of Medicine, Neurology and Neurosurgery Royal Victoria Hospital and Montreal Neurological Institute Montreal, Quebec H3A 1Al Canada

August

Received

lo,1979

SUMMARY: Characterization of somatostatin-like immunoreactivity in rat median eminence and neurohypophysis by gel chromatography yielded two high molecular weight forms in addition to the tetradecapeptide somatostatin. The two larger molecules comprised 5% and 35% of the total tissue immunoreactivity, showed molecular weights of 25000 and 4000 dalton and were both releaseable in vitro in response to depolarizing stimuli. Their characteristic elution volumes remained unchanged after treatment with dithiothreitol or boiling in 8 M urea. Gentle trypsinization of the 25000 dalton molecule resulted in partial conversion into irmnunoreactive material coeluting with the tetradecapeptide somatostatin. Since the antibody employed in these studies is specific for the central and C-terminal portions of the tetradecapeptide somatostatin the present data suggest that both high molecular weight forms represent N-terminal extensions of somatostatin and that the 25000 molecular weight material might represent a prohormone for somatostatin. INTRODUCTION It involves are

is

now well ribosomal

subsequently

a number

of

of reports

have

that of high

suggested

The present

molecular

precursor

(SLI)

with

was undertaken

and neurohypophysis,

weight

enzymatic

somatostatin

been identified study

of secretory

cleavage

the presence

immunoreactivity

yet

the biosynthesis

by specific

the tetradecapeptide

SRIF has not

eminence

synthesis processed

somatostatin-like that

established

tissues

with

(SRIF), certainty

Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

466

tissues

of

greater

than

molecule

for

(3 - 6). SLI in shown

1 Fellow, Medical Research Council of Canada 2 To whom all correspondence should be addressed. Abbreviations: SLI, somatostatin-like immunoreactivity; peptide somatostatin; DTT, dithiothreitol; 25 K, 25000 4000 dalton. 0006-291X/79/180466-07$01.00/0

which

Although

weight

a precursor

to characterize previously

forms

(1,2).

in different molecular

peptides

the rat

to contain

SRIF, dalton;

median

a high

tetradeca4 K,

Vol. 90, No. 2, 1979

concentration

BIOCHEMICAL

of somatostatin

weight

forms

leased

acutely

(7,8).

of SLI are present in response

them may represent

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

We report

in the hypothalamus,

to depolarizing

a precursor

here

stimuli

(prosomatostatin) MATERIALS

that

two high

that

both

and that

forms

at least

molecular are reone of

molecule.

AND METHODS

Tissue Extracts. Adult male Sprague-Dawley rats (150-250 g) were used in the experiments. The animals were kept in a temperature and humidity controlled room and were fed laboratory chow and tap water ad libitum. They were killed by instant decapitation and the median eminence and neurohypophysis were removed as separate fragments and placed in 1 ml 1 N acetic acid at OoC. The tissues were extracted by sonication (Branson sonifier, model 350), the homogenate heated for 5 min in a boiling water bath, rapidly chilled and centrifuged for 10 min at 900 x g (3). The supematant was then analysed by gel chromatography. Gel filtration and radioimmunoassay of somatostatin. Gel filtration was performed on Sephadex G-50 superfine and Sephadex G-100 columns, developped in 6 M urea - 0,05 M phosphate buffer (pH 7;5) or 2 N acetic acid (pH 2,3). The content of immunoreactive somatostatin in each fraction was determined by radioimmunoassay using an antibody directed against the central and Cterminal portions of the SRIF molecule (9). Non-specific interference by urea and acid in the radioimmunoassay was prevented by dilution of the samples prior to assay. The following markers were used to calibrate the columns: Dextran blue, bovine serum albumin, ovalbumin, ribonuclease A, chymotrypsinogen A (Pharmacia, Fine Chemicals, Piscataway, N.J., USA); cytochrome C (Boehringer Mannheim, FRG); e25I] porcine insulin (Novo Industries, Bagsvaerd, Denmark) (10); [125I] pancreatic polypeptide (11); SRIF (Ayerst, Montreal, Canada); 1251 (New England Nuclear, Boston, USA). Elution volumes were expressed as partition coefficients KD where KD = (Ve-Vo)/(Vs-Vo); Ve, elution volume; Vo, void volume; Vs, salt volume. Dithiothreitol and trypsin experiments. Column fractions eluted in 2 N acetic acid containing higher molecular forms of SLI were pooled separately, lyophilized, reconstituted in 0.05 PO4 buffer (pH 7.5) and used for dithiothreitol (DTT) and trypsin experiments. Treatment with DTT was performed by exposing SLI to DTT (loo-fold molar excess) at 50°C under 100% N2 for 3 hr Trypsinization was carried out by treatment of SLI with trypsin (10). (TPCK-trypsin, Worthington Biochemical Corp., Freehold N.J.) at a concentration of 0.1% or 5% w/w at 25oC, pH 8.2 for 1, 10 or 100 min. The reaction was terminated by addition of 1 ug soyabean trypsin inhibitor (Boehringer Mannheim, FRG). Somastatin release experiments. Pools of 5 median eminences or neurohypophysis were incubated for 30 min periods in 1 ml Locke's solution as previously described (7,8). Somatostatin release was stimulated by increasing the K+ concentration in the medium from 5.6 to 56 mM. Medium containing the released somatostatin was chromatographed on Sephadex G 50 columns as described above for the extracts. Statistical of unknown tested for by a t-test parallelism

analysis. To substances and homogeneity by both with the was indicated

check for paralellism between serial dilution slopes SRIF, the residual variances of the two curves were a f-test and the lines were tested for parallelism aid of a computer program (13). Significant nonby a p-value 4 0.05 in the t-test.

467

Vol. 90, No. 2, 1979

A o--

D

BIOCHEMICAL

c ---

IPP

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

125,

B 5-

SEF

D 33.

C 35

35

p40i3 54

B G $

>

s 37~

45 0

05

lx)

0

025

1.0 KD

KD

of immunoreactive somatostatin in tissue extracts Fig. 1. Characterization and incubation media by Sephadex G-50 (superfine) gel chromatography. A 1.15x56cm column was eluted with 6 M urea in 0.05 M PO4 buffer, pH 7.5. KD = partition coeff cient. Molecular wei ht markers: D, dextran blue; C, cytochrome C; I, if 25Sinsulin; PP, Id+ ancreatic polypeptide; SRIF, synthetic somatostatin (tetradecapeptide); *51, radioactive iodine. A : Extract of neurohypophysis. B : Extract of median eminence. c: Incubation medium containing im=oreactive somatostatin released in vitro from neurohypophysis following stimulation by K+ (56 mM). D : Incubation medium containing immunoreactive somatostatin released in vitro from median eminence following stimulation by K+ (56 mM).

RESULTS Sephadex hypophysis

showed

Identical

elution

6 M urea

of extracts

peaks

patterns acid.

material

eluting I accounted

peak II

innnunoreactivity. a Sephadex

G-100

with for

obtained 60% of

column

with

KD of 0.11 approximately

468

markers

about weights

1A and B). in either coeluted

higher

and 0.43 total

and neuro-

(fig.

developed

comprised

I SLI)

and represented

with

eminence

immunoreactivity

5% of the

of the molecular

calibrated

columns

peaks (peak

median somatostatin

the total

The additional

was more prominent Estimation

of both

of inununoreactive

were

SRIF at KD 0.73.

SLI in peak whereas

three

or 2 N acetic

synthetic weight

G-50 filtration

with

molecular

(peak

II

SLI).

imrnunoreactivity 35% of the

total

of the two peaks

of known molecular

weight

using

Vol. 90, No. 2, 1979

BIOCHEMICAL

2

1

AND BIOPHYSICAL

5

10

20

Wefght

x 10e3

Molecular

RESEARCH COMMUNICATIONS

50

100

Fig. 2. Molecular weight calibration curve using Sephadex G-100. (0.9 x 58 cm column; flow rate 0.15 ml/min; hydrostatic pressure, eluting buffer, 6 M urea in 0.05 M PO4 buffer, pH 7.5).

showed

apparent

molecular

and II

SLI respectively The in vitro eminence

over

levels

specific Gel with

KD values In order dilutions

inhibiton

material

and peak

II

released

I and peak

two forms to SRIF. of either

SLI with

trypsin

of

II

for

peak I

SLI were

SLI did

boiling

in

I or peak for

469

the

incubation

3 peaks

assayed.

'big'

5% (w/w)

respectively The release

II

weight

yield

8 M urea SLI.

10 min led

medium.

(fig

1C and D)

materials,

The resulting

SRIF (fig not

was

extracts.

molecular

synthetic

peak

in

the tissue

with

Also

and 5 fold

yielded

the higher

parallelism

position

of Ca"

obtained

of peak

of the

for

from neurohypophysis

K+ (56 mM) stimulation.

immunoreactivity

to those

showed

9 fold

on the presence

characterize

corresponding

elution

to high

further

curves

ment of either

the

'to

and 4000 dalton

somatostatin

was increased

in response

identical

dalton

2).

fragments

of the

of 25000

of irmnunoreactive

and was dependent

filtration

serial

(fig

release

and median basal

weights

100 cmH20;

3).

DTT treat-

any immunoreactive for

5 min did

Incubation to a complete

not

of peak loss

alter I

of

Vol. 90, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL

pg SRIF

. 3. Fig. SLI with

Parallelism synthetic 21

between serial SRIF. (Peak III:

dilution material

or

RESEARCH COMMUNICATIONS

ui sample

slopes of peak I, II and III coeluting with SRIF). SRIF

1

Fig. 4. Elution profile of peak I SLI after gentle trypsinization (O.l%(w/w) 100 min) on Sephadex G-50 (superfine). Elution buffer 6M urea in 0.05 M PO4 buffer, pH 7.5)

immunoreactivity. for

100 min),the

immunoreactive immunoreactivity represent the

Using bulk species with

an intermediate

tetradecapeptide

more of peak

gentle

however

I SLI was converted

coeluting KU 0.30

conditions

with

SRIF

and 0.81

breakdown

were

product

SRIF respectively.

(fig also

470

to a lower 4).

0.1% (w/w) molecular

Two additional

obtained.

of peak

Treatment

(trypsin

These

weight

peaks probably

I SLI and a fragment

of peak

II

of

SLI with

of the

BIOCHEMICAL

Vol. 90, No. 2, 1979

low concentration 1.0)

which

presence

of trypsin

overlapped of both

led

AND BIOPHYSICAL

to a broad

the elution

volume

SRIF and other

peak

RESEARCH COMMUNICATIONS

of immunoreactivity

of SRIF (KD 0.73)

immunoreactive

(KD 0.47-

suggesting

the

fragments.

DISCUSSION The existence

of high

has been previously present

study

demonstrated

we have

and neurohypophysial molecular

weights

approximately studies

to those

our gel

of conversion

of SRIF it

studies

would

SRIF connected relationship

obtained

with

comparable

the

in

precursors

(1,2),

via to SRIF is

a N-terminal uncertain

471

the postthe present with

of the 25 K mole-

of the antibody and C-terminal

a N-terminal amino

acid

residues.

less

clear

from

the present

extension

of SRIF although

of the

emregions

extension

basic

because

trypsinization.

8 M urea,

coeluting

a role

the middle is

re-

or polymerized

implicated

for

denatur-

molecules

DTT or boiling

specificity

25 K molecule

tetradecapeptide

both

aggregated

evidence

the

of SRIF is

from

strongly

to material

toward

too may represent

as a precursor

hormone

Since

under

with

protein-bound,

directed

that

for incubation

released

findings

and since

treatment

provides

of the 4 K molecule It

acid)

25 K SLI peptide

SRIF.

are

with

account

the in vitro

forms

performed

enzymes have been

is

appear to the

were

of peptide

for

eminence

(1,2,14).

after

represent

trypsinization

in these

studies.

even

together

stimulus,

or 2 N acetic

of the

as a prohormone

ployed

cells studies

modification

SRIF by gentle

role

they

weight

In the

of two polypeptides

Furthermore

molecular

(3-6).

SLI in median

to consist

to a depolarizing

trypsin-like

translational

cule

high

intact

Because

finding

both

'big'

of 25 K and 4 K which

filtration

that

this it

of SLI in the hypothalamus

of investigators

immunoreactivity.

(6 M urea

unlikely

SRIF.

region

response

essentially

is

and shown

in non-neural

conditions

mained it

in

reported

Since ating

extracts

that

forms

by a number characterized

40% of total

tissues

weight

further

in the

revealed

these

molecular

inconclusive

of The

its results

Vol. 90, No. 2, 1979

Further coupled help

with

to clarify

possibly

other

studies

BIOCHEMICAL

of the biosynthesis

structural the

AND BIOPHYSICAL

analysis role

hypothalamic

of SRIF by hypothalamic

of the high

of these

RESEARCH COMMUNICATIONS

molecules

molecular as precursors

weight for

neurons forms

should

SRIF and

peptides. ACKNOWLEDGEMENT

The authors wish to thank work was supported by the USPH grant No AM 21373.

Mr. T. Wheatley for Canadian and Quebec

technical Medical

This assistance. Research Councils and

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Steiner, D.F., Kemmler, W., Tager, H.S., and Peterson, J.D. (1974) Fed. Proc. 33, 2106-2115. Habener, J.F., Kemper, B.W., Rich, A., and Potts, J.T.,jr. (1977) Rec.Prog.Horm.Res. 33,249-299. 102, 523-530. Patel, Y.C. and Reichlin, S. (1978) Endocrinology Schally, A.V., DuPont, A., Arimura, A., Redding, T.W., Nishi, N., Linthicum, G.L., and Schlesinger, D.H. (1976) Biochemistry 15, 509-514. Rorstad, O., Epelbaum, J., Brazeau, P., and Martin, J.B. (1977) Clin.Res. 25, 684A. Spiess, J., Villarreal, J., and Vale, W. (1979) The Endocrine Society, 61st annual meeting, program and abstracts, p. 146. Patel, Y.C., Zingg, H.H., and Dreifuss, J.J. (1977) Nature 267, 852-853. Patel, Y.C., Zingg, H.H.,and Dreifuss, J.J. (1978) Metabolism 27, 1243-1245. Vale, W., Ling, N., Rivier, J., Villarreal, J., Rivier, C., Douglas, C., and Brown, M. (1976) Metabolism 25, suppl.l., 1491-1494. D.M. (1971) Biochem.Biophys. Freychet, P., Roth, J., and Neville, Res.Commun., 43, 400-408. Chance, R.E., Moon, N.E., and Johnson, M.G. (1979) Methods of Hormone Research, 2nd edition , pp.657-672, Academic Press, New York. Konigsberg, W. (1972) Methods Enzymol. 25, pp 185-188, Academic Press, New York. Faden, V.B., and Rodbard, D. (1975) Radioimmunoassay Data Processing: Listing and Documentation, 3rd edition, vol. 1. National Technical Information Service, U.S.Dep.of Commerce, Springfield, VA, USA. Allen, R.G., Herbert, E., Hinman, M., Shibua, H., and Pert, C.B. (1978) Proc.Natl.Acad.Sci. USA 75, 4972-4976.

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