Hormonal stimulation of cAMP-dependent protein kinase in rat pancreas

Vol.

95,

No.

2, 1980

July

31,

1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

553-561

Pages

HORMONAL STIMULATION 0. Holian,

OF CAMP-DEPENDENT PROTEIN KINASE IN RAT PANCREAS

Ph.D.,

C. T. Bombeck,

M.D.,

Lloyd

M. Nyhus,

M. D.

From the Department of Surgery, University of Illinois at the Medical Center, The Abraham Lincoln School of Medicine, Chicago, Illinois

Received

June 6,198O SUMMARY

Rat pancreas possesses a CAMP-dependent protein kinase enzyme which is inhibited by the protein kinase inhibitor from bovine heart. Incubation of rat pancreas tissue in the presence of secretin and CCK-PZ resulted in an increase of intracellular CAMP and activation of the CAMP-dependent protein kinase. CCK-OP, a pure synthetic analog of CCK-PZ, did not stimulate cellular CAMP production or protein kinase activation. There was correlation between the ability of a given dose of hormone to stimulate CAMP formation and activate the protein kinase enzyme. It is believed that the stimulatory effect of CCK-PZ is due to secretin contamination of this hormonal preparation. Many of theeffects tissues

are

dent

protein

with

the

of cyclic

believed

to be mediated

kinase

(1).

regulatory The catalytic

resulting

in an intracellular (3)

can be studied

in vitro

exocrine

CAMP levels agents

pancreas.

in tissue

CCK has been guinea

using

cells

pig

levels

protein

the

activation

slices.

as the

second

and vasoactive

isolated

from

guinea

(CCK) had no effect of cyclic

kinase

(2).

of protein

Soderling

kinase

of hormonally

intestinal pigs

in the

by CAMP

stimu-

peptide

(4,5).

monophosphate

cyclase

catalytic

substrate,

increased

Cholinergic

on CAMP levels,

guanosine

adenylyl

by combining

an active

action

messenger

mammalian

of CAMP-depen-

appropriate

of hormonal

tissue

shown to stimulate

but caused (cGMP).

pancreas

an

However

of rat

and

(6,8).

The aim of the present dent

protein

enzyme and liberating

hormonal

Secretin

(cAMP)in

in the activity

phosphorylates

that

and cholecystokinin

increase

of the

implicated

in acinar

by changes

expression

showed

monophosphate

AMP activates

subunit

and associates

lated

Cyclic

subunit

subunit.

CAMP has been

adenosine

kinase

work

by stimulation

was to study

the activation

of endogenous

CAMP formation

of CAMP-depenin the

presence

0006-291X/80/140553-09$01.00/0

553

Copyright Q 1980 b-v Academic Press, Inc. All rrghts of reproduction in an-v form reserved.

Vol.

95, No.

2, 1980

of hormones. with

BIOCHEMICAL

It was of special

interest

cholecystokinin-pancreozymin

cholecystokinin

AND

BIOPHYSICAL

RESEARCH

to compare

the effects

(CCK-PZ),

and a synthetic

COMMUNICATIONS

of secretin

octapeptide

of

(CCK-OP). MATERIALS AND METHODS

Tissue Incubation. Pancreata were obtained from male Sprague-Dawley rats weighing 150 to 200 gm fed on a standard meal ad libitum. Prior to the experiThe rats were killed by a blow to the ment, the rats were fasted for 48 hours. head; the pancreas quickly removed, dissected free from fat and mesentery and used immediately. The pancreas was cut into small pieces and placed in Ringer's bicarbonate buffer, pH 7.4, containing 1 mg/ml glucose and 0.5 mM 3-isobutylThe tissue was preincubated at 37O C for ten minutes l-methyl xanthine (IMX). with mild shaking. At the end of the preincubation period, the tissue was transferred to fresh buffer containing the appropriate hormones and incubated for additional ten minutes at 37O C. Two to three pieces of tissue, approximately in 5 ml of buffer. At the end of the incubation 2 to 4 mm2, were incubated period, the tissue was quickly removed, frozen in liquid nitrogen, and subsequently stored under liquid nitrogen. Enzyme Preparation. Frozen tissue was pulverized in liquid nitrogen and the pulverized powder homogenized in ten volumes of 10 mM potassium phosphate buffer, pH 6.5, containing 10 mM ethylene diamine tetraacetate (EDTA) and l165cmM IMX. The homogenate was centrifuged at 27,000 x g for 20 minutes at The supernatant fraction was used as the enzyme preparation. Protein kinase assay was performed imnediately after enzyme preparation. Protein Kinase Assay. The reaction was started by adding 5 to 20 ~1 of the enzyme preparation to 50 ~1 of a solution containing 17 mM potassium phosphate buffer,3$ H 6.8, 10 mg/ml histone (Sigma type II-A), 6 mM magnesium acetate, 0.33 mM ATP-y p (20 to 50 counts per minute/pmole) in the presence and in the absence of 2 PM CAMP. The reaction mixture was incubated at 30o C for 5 minutes and the reaction terminated by addition of 1 ml of ice cold 10% trichloroacetic acid (TCA). The formed precipitate was collected by filtration on Whatman GFlC glass fiber filters, washed four times with 2 ml portions of ice cold 10% TCA, and counted in 5 ml of Bray's solution (9) in a Packard Tri-Carb liquid scintillation counter. The protein kinase activity was expressed as pmoles of 32p incorporated into histone per milligram of protein per minute. The protein kinase activity ratio was expressed as the ratio of protein kinase activity in the absence of added CAMP to protein kinase tivity in the presence of 2 PM CAMP. In each assay nons ecific binding of 35 p to histone was determined by measuring the amount of 3! P bound to denatured enzyme. The value obtained was used as zero time incubation, and was subtracted from all other values. CAMP Assay. Frozen tissue, previously incubated with and without hormones, was homogenized in 3 ml of ice cold 6% TCA and 6000 dpm of 3H-cAMP were added to determine recovery. The homogenate was centrifuged at 10,000 x g for 30 minutes at 4O C. The supernatant fraction was applied to a Dowex 50-H+ form column, 100 to 200 mesh, 0.9 x 15 cm, which had been equilibrated with O.lN HCl. The column was eluted with O.lN HCl, the CAMP elution profile determined, and the fraction containing CAMP was collected and lyophilized to dryness. The lyophilized fraction was resuspended in 1 ml of water and the concentration of CAMP was determined against a standard curve by the method of Gilman (10). The CAMP values obtained were corrected for recovery and expressed as pmoles of CAMP per milligram of protein. Protein Determination. of Lowry (11).

Protein

concentration

554

was determined

by the method

Vol.

95, No.

2, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Materials. Type II-A histone, CAMP-dependent protein kinase inhibitor from bovine heart, CAMP, ATP, IMX, and binding protein were purchased from porcine preparation of secretin and the Sigma Chemical Co. The natural CCK-PZ were purchased from the Karolinska Institute, Stockholm, Sweden; this supplier states that each ampule containing 75 U of CCK-PZ also contains 0.7 U of secretin. CCK-OP wgs a gift of the Squibb Institute for Medical Research; ATP-~32P and CAMP- H were purchased from the New England Nuclear Corporation. All other chemicals used were of the highest purity commercially available. RESULTS Control caused

Studies.

of exogenous

a concentration-dependent

histone

(Fig.

1).

increase

Activation

CAMP concentration tion

Addition

in pmoles

of protein

of 0.05

CAMP to the

kinase

PM and was optimal

reaction

mixture

of 32P incorporated

was already

into

apparent

at 0.4 MM, when the

at a concentra-

of the enzyme was 200 ug protein. The effects

kinase

activity

reaction

are

shown

was carried

centration It

of protein

between

out

probably holoenzyme.

effect caused

in Fig. for

five

(12,13)

In all

minutes

that

on the protein by stabilization

Increasing

and time

2.

100 to 500 vg per

has been shown

stabilizing

concentration

subsequent

at 30'

on protein

experiments

C maintaining

the

protein

con-

tube. in some tissues

kinase

activity

of dissociation

concentrations

addition ratio. of the

of NaCl present

CAMP

Effect of was incubated cAMP concentrations, point was determined of two others.

of incubation

This protein during

effect

is

kinase tissue

(PM)

CAMP on protein kinase activity. for 5 minutes at 30° C in the and protein kinase activity was in triplicate, and this experiment

555

of NaCl has a

The enzyme (200 vg presence of varying determined. Each is representative

homo-

Vol.

95,

No.

2, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

60.

I -0

50 MINUTES

100

OF INCUBATION

pq

I 200

I 800

400

PROTEIN

Effects of incubation time and protein concentration on the F$S%Ginase activity In the top curve 100 ug protein were incubated for different times at iO" C in the presence and absence of 2 UM CAMP. In the bottom curve varying amounts of protein were incubated for 5 minutes at 300 C in the presence and absence of 2 LIM CAMP. Each point was determined in triplicate and each experiment is representative of two others. The dashed line represents values in the absence of CAMP; solid line represents values in the presence of 2 LIM CAMP.

genization

caused

indicating

an

to

dissociation

in

the

by

of

the

activity

component

kinase

portion

protein,

Effects.

As

with

a secretin

of

ratio

of

holoenzyme.

the

the

NaCl

protein

kinase

inhibitor

protein

kinase

shown

secretin

was

no

further

to

1o-4

M.

in

tissue

of

CAMP

of were

increase The

CAMP a response

in

natural level

(Fig.3),

enzyme was

kinase from

probably

due

therefore

activity

bovine

activity

of

the

Fig.

5,

omitted

was

heart

inhibited

(Fig.

approached

cAMP

higher same

CAMP

CCK-PZ.

10m7

M.

The

level

4).

that

nine

times

when

in

concentrations of

magnitude

556

was

the

At the

tissue

rose

already

apparent

basal

was level.

concentration

CCK-PZ of

the

concentration

secretin of

in

increase

secretin

preparation

order

levels

When

approximately

porcine but

in or

concentration

levels

produce

catalytic

protein

protein

incubation

tissue

kinase

CAMP.

Hormonal after

protein

experiments.

inhibitor of

in the

cAMP-stimulated

of

absence

at

in

a CAMP-dependent

80 pg

increase

increase

following The

an

also

CCK-PZ as

caused were

obtained

There was

raised

an

increase

required with

low5

to secretin.

M,

Vol.

95, No.

03

BIOCHEMICAL

2, 1980

B

05

’

NaCl

Figure 3. centrations The enzyme 5 minutes experiment

20

IO

AND

zr; 04

’

30

BIOPHYSICAL

40

(M)

RESEARCH

PROTEIN

KINASE

COMMUNICATIONS

INHIBITOR

(pig

protein)

Effect of NaCl on protein kinase activity ratio. Varying conof NaCl were present during homogenization of the tissue. was incubated in the presence and absence of 2 nM CAMP for Each point was determined in triplicate, and this at 30° C. is representative of two others.

Figure 4. Effect of bovine heart protein kinase inhibitor on protein kinase activity. The enzyme (47 ug protein) was incubated with varying concentrations of the protein kinase inhibitor in the presence of 2 nM CAMP for 5 minutes at 30° C. The protein kinase activity in the absence of 2 uM CAMP was the same as that in the presence of 75 ng protein of the Protein kinase activity is expressed as per protein kinase inhibitor. cent of the activity obtained with 2 nM CAMP in the absence of protein kinase inhibitor. The

synthetic

carboxy-terminal

cant

changes

were

the

same

octapeptide

CAMP tissue

in as

levels.

At

(CCK-OP) 10 -4

did

M CCK-OP,tissue

not

produce CAMP

signifilevels

controls. ‘30 80

r-

SECRETIN

HORMONE

CONCENTRATION(M)

Figure 5. Effect of hormone concentration on CAMP levels. The tissue was prelncubated with buffer alone for 10 minutes and then incubated with hormones for additional 10 minutes. Each point is a mean t s.e. of three separate experiments, and each experimental value was obtained from quadruplicate tissue samples.

557

Vol.

95. No.

2, 1980

BIOCHEMICAL

I

Table.

EFFECT

OF HORMONES Protein

Hormone Concentration

AND

(M)

BIOPHYSICAL

ON PROTEIN

Kinase

RESEARCH

ACTIVITY RATIO

KINASE

Activity

COMMUNICATIONS

Ratio

(-cAMP/+cAMP)

Secretin

CCK-PZ

CCK-OP

0.188 -t .006

0.188 + .006

0.245 f .017

1O-8 -7 10

0.220

0.198 f .033

0.274 + .019

0.271 T .OOO

0.233 t .012

0.252 + .009

lo-6

0.315 k .016

0.245 f .022

0.288 T .029

10-5

0.419 ?- .OlO

0.300 t .036

0.293 t .025

lo-4

0.482 ?r .OOO

0.355 + .036

0.273 i .029

0

*

.OlO

Tissue was preincubated with buffer alone for 10 minutes, and then incubated with hormones for additional 10 minutes. Protein kinase Each activity ratio was determined as described under methods. point is a mean + s.e. of three separate experiments, and each experimental value was obtained from quadruplicate tissue samples.

Incubation

of pancreatic

concentration-dependent The ratio

increase

increased

.OOO in the

from

presence

CCK-PZ produced

The synthetic

kinase

activity

of 10

caused

by animal

with

in the + 0.066

-4

secretin protein

when tested

kinase

produce

between

in the CCK-OP experiments and experimental

CCK-PZ produced activity

any added

Incubation

in the protein not

or kinase

without

M secretin.

CCK-OP did

ratio

ratio

0.188

an increase

.036.

activity

tissue

ratio

hormone

of tissue activity

with

ratio

I).

+

10m4 M

to 0.355

change

10m8 to 10s4 M.

The higher

be explained

(Table

to 0.482

any significant

cannot

a

+

in protein basal

but may have been

variations. DISCUSSION

Stimulation changes fluid

of exocrine

in tissue

levels

and bicarbonate

system

(E-20).

of CAMP during stimulation

pancreatic

of CAMP (14). secretion

On the other CCK-stimulated of adenylyl

administration

of the

of increased

CAMP levels

secretion

are

is

mediated

hand,

although

has been shown that through data

enzyme secretion

cyclase hormone

It

activity

the point

(4),some

and increased

(8,21-24). believed

is thought

The metabolic to be expressed

558

to be related

to

stimulation

of

CAMP-adenylyl

cyclase

to the noninvolvement reports cellular

have shown CAMP after

and functional through

the

effects

activation

Vol.

95, No.

2, 1980

BIOCHEMICAL

of CAMP-dependent kinase

protein

cells

confirmed

that

isolated the

tains

a protein

study

demonstrates

using

an in vitro

enzyme which

hormonal

There hormones

in elevating

enzyme.

When it

between tissue

intracellular

CAMP.

subsequent

stimulation

assumed

enzyme This

of fluid

is

responsible for

known

that

secretin. the

of secretin

weights

of the

CCK-PZ preparation

This

con-

protein

kinase

secretin

buffer levels

with

secret

of CAMP.

as was CCK-PZ.

the degree

stimulation

In

analog

only

by stimulation the

of

formed

of CAMP and protein

contains

can account

for

CCK-PZ preparation.

559

that enzyme

kinase

production

degree

two different the

hormonal

secretion

is

activation,

It is

Taking

into

that

and each

2 x 10e3 doles of stimulation

of

with

(see Materials)

can be calculated

approximately the

the

of

activates

kinase

by secretin.

in the CCK-PZ preparation it

secretin

of enzyme secretion.

contaminated

two hormones,

with

indicate

of pancreatic

stimulants

that

a protein

The results

protein

production

in increased

CAMP activates

equipotent

kinase

CAMP-dependent

concept

resulting

stimulation

of the

the protein

of endogenous

current

pancreas,

is

of potency

of the

(CCK-PZ and CCK-OP)

CCK-PZ preparation

amount

the

the stimulation

CCK-PZ and CCK-OP are

mMole of the

pancreas

has

In additian;this

in tissue

and activating

secretion.

for

since

the molecular

and

Our study

of rat

a stimulant

between levels

that

newly

moiety

the

(25-27)

CAMP levels.

in the

of cholecystokinin

account

rats

(28).

bicarbonate

increase

strengthen

preparations

the

pigs

by CAMP.

in Ringer's

relationship

findings

cyclase

responsible

from

protein

-4 M CCK-OP, the synthetic to 10

CAMP tissue

is

CAMP, the present

not

of guinea

as potent

low8

can be achieved

which

of pancreata

of the CAMP-dependent

tissue

loo-fold

was a general

an adenylyl

of a CAMP-dependent

stimulated

a dose-dependent

not elevate

activity

is

COMMUNICATIONS

system.

ranges

did

RESEARCH

of the homogenate

stimulation

was approximately

CCK-PZ,

kinase

fraction

of pancreatic

concentration

The presence

the pancreata

supernatant

or CCK-PZ produced

Secretin

BIOPHYSICAL

in homogenates from

kinase

Incubation tin

kinases.

has been demonstrated

in acinar

AND

found

of with

Vol.

95, No.

2. 1980

Complete is

possible

activation that

of the protein resulting

reassociation

activity

activity

ratio findings

pancreatic

cholecystokinin

both confirm

kinase

during

RESEARCH

COMMUNICATIONS

catalytic

tissue

and regulatory

homogenization

subunits

and assay,

Preliminary

experiments

indicate

tissue

homogenization

results

in a higher

that

enzyme secretion,

CAMP is

and presence

of hormones.

not

during

involved

and stimulation to secretin

It

enzyme was not achieved.

ratio.

in the absence

may be attributed

BIOPHYSICAL

of the

enzyme occurs

of 0.5 M NaCl during

the present lated

kinase

AND

of the protein

a rapid

in a lower

presence kinase

BIOCHEMICAL

of adenylyl

contamination

that protein In summary,

hormonally cyclase of this

stimuby

hormonal

preparation. ACKNOWLEDGEMENT We wish to thank Mrs. Cathy Ruiz for her expert technical This work was supported in part by PHS-AM 20838-01 grant from Institutes of Health.

assistance. the National

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:: i: 5. 6. 7. 8. 9. :::

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Park, C.R. (1973) J. Biol. Chem. 248, Soderling, T.R., 1813-1821. Keely, S.L.,Soderling, T.R., et al. (1975) in Advances in Corbin, J.D., Cyclic Nucleotide Research (Drummond,G.I., Greengard, P., Robison, ,G.A. Eds.) Vol. 5, p 265-279 Raven Press, New York. Kimberg, D.V. (1974) Gastroenterology 67, 1023-1064. Tompkins, R.K., Kuchenbecker, S.L. (1973) J. Surg. Res. x,172-176. G. (1974) Life Sci. I& 631-639. Heisler, S., Grondin, G., Forget, Deschodt-Lanckman, M., Robberecht, P., DeNeef, P., et al. (1975) Gastroenterology 68, 318-325. Lemon, M.J.C., Bhoola, K.D. (1975) Biochim. et Biophys. Acta 385,101-113. Domschke, S., Konturek, S.J.,Domschke, W., et al. (1975) Proc. Sot. Exp. Biol. Med. 150, 773-779. Domschke, S., Domschke, W.,Konturek, S., et al. (1976) Gastroenterology

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223,

Biol. ~,253-257. Biochim. et Biophys. Biochim.

et

Biochem.

Biophys.

Biochimie,

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