Partial agonist activity of the bombesin-receptor antagonist [Leu14-ψ-CH2-NH-Leu13]-bombesin in frog peptic cells

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

December 30, 1988

Pages 1154-115~'

PARTIAL AC.~NIST._I,ACTIVITY OF THE BOMBESIN-RECEPTOR ANTAGONIST [Leu'ZV~-CH2-NH-Leu'3]-BOMBESIN IN FROG PEPTIC CELLS Kenneth E.J. Dickinson, Naomi Uemura, M. Chandra Sekar, Huey B. McDaniel, Wayne Anderson, David H. Coy* and Basil I. Hirschowitz Division

of Gastroenterology,

University

of Alabama

at

Birmingham,

A L 35294

*Peptide Research Labs, Tulane University School of Medicine, N e w Orleans, LA 70112 Received November ii, 1988

14 13 The pseudopeptide [Leu - ~ - C H 2 N H - L e u ]-bombesin inhibited 1251G R P binding to membrane preparations of frog cerebrum and peptic cells, r%t~cerebral cortex and pancreas with IC50's of 44-250 nM (using 180 p M - - ~ I - G R P ) . It was unable to stimulate amylase release from rat pancreatic acini, but antagonized competitively B B stimulated amylase release with an IC_ 0 of 130 nM. By contrast the pseudopeptide stimulated pepsinogen ~ecretion from frog esophageal peptic cells with an efficacy relative to bombesin of 36%, and with an EC_^ of 30 nM. By virtue of its partial agonist activity it inhibited subn~aUximal BB stimulated..responses to a~level equal to the pseudopeptide alone. Thus [LeuJ4-~-CH2NH-LeuI~]-BB differentiates certain B B receptors by exhibiting selective intrinsic efficacy. © 198, Aoademio P..... ~nc.

Studies of bombesin's factor in human

neoplasms

selective antagonists.

action as a mitogen

(I) and

autocrine growth

(2) require the development

of high affinity,

Early attempts were based on analogues of substance

P(3) and D-[Phe 12] analogues of bombesin and

relatively low affinity were

synthesized

pseudopeptide

(4), although lack of specificity

significant

disadvantages.

[Leu14-~-CH2NH-Leu13]-BB

The

(LL-BB)

recently exhibited

no detectable amylase releasing activity from guinea pig pancreatic acini and antagonized

a number

of in vitro B B

stimulated

responses

with

IC50's of

18-35 nM (5). We have been studying pepsinogen and

have

pepsinogen LL-BB

reported

the

secretion

presence

(6).

The

release from frog esophageal mucosa

of B B present

receptors report

which

compares

are coupled

the activity of

in the amphibian peptic cell system with that in rat pancreas.

binding techniques we also compare its potency for B B and peripheral tissues. 0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

1154

to

Using

receptors in C.N.S.

Vol. 157, No. 3, 1 9 8 8

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS AND METHODS Synthesis of [Leu13-~-CH~NH-Leu14]-BB: Solid phase syntheses were performed using standard techr~iques described previously ( 5 , 7 ) . Membrane Preparation: Brain tissue was homogenized in 10 vols (or 10 ml for peptic cells and pancreatic acini) of 50 mM Tris HCI, 5 mM MgCI~ pH 7.4 using a Potter Elvejehm homogenizer. The homogenate was centriffJged at 6 0 0 g for 10 rain, the pellet discarded and the supernatant was recentrifuged at 40,000g for 20 rain. The resultant pellet was washed thrice and suspended in buffer at a protein concentration of I-3 mglml. 1251-GRP Bindincj: Membranes were incubated with 1251-GRP (150-200 pM] in 50 mM T r i s HCI, 5 mM MgCI~ b u f f e r containing 0.2% BSA, 1 mglml Ip~itracin in a 0.1 ml total volume Zfor I hour at 22°C. Bound and free - - - I - G R P were separated by vacuum f i l t r a t i o n through Whatman GFIC f i l ters, followed by washing with 3x5 ml of b u f f e r . Filters were presoaked in 0.2% polyethyleneimine and f i l t e r blanks were I-2% of added radioligand. Bound radioligand1~,~as counted in a Isoflex gamma counter at 75-80% e f f i ciency. Specific "'~I-GRP binding was defined using I ~M bombesin and protein concentrations were choselr~,such that < 15% of added radioligand was bound at equilibrium. KD's o f - ~ I - G R P for rat brain and pancreas membranes were 1.1 nM. Specific binding represented 78% ( r a t pancreas); 62% ( r a t cerebral c o r t e x ) ; 71% (frog cerebrum) and 44% (frog peptic cells) of total binding. IC50 and Hill slope factors were calculated from Hill plots. Protein was determined using Pierce BCA protein assay (Pierce, Rockford, IL). Amylase Release from Rat Pancreatic Acini: Rat pancreatic acini were prepared by collagenase digestion as described previously (8). Acini were incubated with secretagogues for 30 rain at 37°C, centrifuged at 13,000 g and aliquots of supernatant assayed for amylase a c t i v i t y (9). Pepsinogen Release from Isolated Peptic Cells: Peptic cells were isolated from frog esophageal mucosa using the procedures described by Matsumoto et al (10), with the omission of EGTA f r o m the media used to separate cells from glands. Peptic cells were incubated for 30 min at 22°C with secretagogue and pepsinogen released into the medium was assayed using a sensitive automated acidified hemoglobin digestion method (11).

RESULTS AND DISCUSSION Table I

compares the a b i l i t y of BB and

LL-BB

to i n h i b i t

1251-GRP

binding to membranes of frog and rat tissue. Bombesin exhibited similar IC50 values ( 0 . 8 - I . 6 frog peptic cells,

nM) for receptors of

rat cerebral cortex and pancreas, and showed reduced

potency for frog cerebral receptors (4.6 nM).

LL-BB had similar a f f i n i t i e s

for receptors on frog peptic cells and rat pancreas and reduced potencies for rat cerebral cortex and frog cerebral BB receptors. observed

in

the

Hill

slope

factors

of

competition

glandular tissue where slopes approached

unity,

Differences were

curves

and brain

generated

in

tissue where

slopes were s i g n i f i c a n t l y < I . Fig. I shows the effect of LL-BB on basal and BB stimulated amylase release from rat pancreatic acini. dependently

(Fig.

Bombesin stimulated amylase release dose

2) with an EC50 of 0.3 nM. 1155

The pseudopeptide alone

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table I : IC50 values and Hill slope factors (nil) of BB and LL-BB were calculated from competition curves. Values represents means (±SE) of experiments performed 4-7 times. Tissue Frog Cerebrum Frog peptic cells Rat cerebral cortex Rat pancreas

produced

4.6(±0.7) 1.6(±0.24) 0.77(±0.12) 1.1(±0.05)

0.82(±0.04) 0.92(±0.05) 0.79(±0.03) 0.93(±0.02)

I).

BB

inhibition of B B

Fig. 3 shows peptic cells.

lack

BB

and

of

(I0-9M)

stimulation,

stimulated

the effects of bombesin stimulated pepsinogen

with

an

overall

intrinsic activity

ECs0

with an IC50 of 130 nM

secretion was

and

competitive

and

activity

with

LL-BB

an

LL-BB

on

of I nM.

was

able

concentrations of BB. I nM B B

of 30 to

Thus

nM.

By

In marked LL-BB

These

results with

indicate

BB

that

receptors

virtue

inhibit secretion

10 pM L L - B B

LL-BB

stimulated

and

to its

pepsinogen

was 36% of that

of this partial agonist

stimulated

by

submaximal secretion by

alone (Fig. 4).

exhibited

of frog

contrast

inhibited pepsinogen

to a level of that of pseudopeptide

interaction

frog esophageal

secretion over a large concentration

in rat pancreas,

EC50

as shown

(Fig. 2).

secretion from peptic cells with an intrinsic efficacy which of BB,

nH

204(±33) 0.79(±0.05) 44(±5) 0.91(±0.09) 250(±50) 0.81(±0.08) 106(±10) 0.94(±0.05)

by the parallel shift of the B B dose response curve

range,

LL-BB

no statistically significant stimulation of amylase release (p < 0.05)

but it potently antagonized (Fig.

IC50 (nM) nH

BB

significant

rat tissue.

potency

for

values

for

IC50

--~ 100 I

•~-

60

~

so

'~

40

~

4o

"~

20

~"

20

I

Y/?

0 I

I

I

I

I

I

I

I

I

I

I

I

I

9

8

7

6

5

12

11

10

9

8

7

6

5

-log [LL-BB], M

G

@

-log [BB], M

Fi9. It:atConcentration dependence of LL-BB alone (6~ on amylase release from pancreatic acini and inhibition of BB (10-~M) stimulated amylase release by LL-BB (e). Fi9. 2: Dose-response curves dof BB-stimula_t~d amylase release in the absence (1) and presence of 10-vM (o) and 10 ~M (A) LL-BB. (Results are mean curves of experiments performed 3-5 times. SE's were < 10%.) 1156

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

100 5

o

80

~, '

4

60

b

:

40

n

i

90

)

o t

I

i

I

I

i

i

i

12

11

10

9

8

7

6

5

-log [ s e c r e t a g o g u e ] ,

L-BB

BB

M

BB+ LL-BB

®

Fi9. 3: Dose-response curves for BB (o) and LL-BB (&) stimulated pepsinogen secretion from frog esophageal peptic cells. Results are mean curves of experiments performed 4-8 times and SE's were < 10%.

~

: Pepsinogen r~cretion of peptic cells in response to half maximal BB ), LL-BB (10-M), and the combination. Results show mean (_+ SE) of 3 experiments.

inhibition of 1251-GRP to those obtained (5). BB was

However,

binding

to peptic cell and

for its interaction whereas

receptors on guinea

LL-BB

with guinea

exhibited

pig and

examined

were

pig pancreas

(KD=60

rat pancreas,

function

receptors.

the ability of L L - B B

suggesting

as an some

antagonist

and

partial agonist

may

be able to activate certain

Recently

to antagonize

not

others

(e.g.

pancreas),

induced

receptors

where

been

in the

receptors.

(e.g.

it has

Cowan

at

et al

hypothermic

The pseudopeptide

scratching

activity at C N S BB

nM)

and Swiss 3T3 cells (5), it

scratch responses to ICV B B administration in rats. not

similar

little or no intrinsic efficacy

a partial agonist at peptic cell B B

(12) have

rat pancreas

and did

latter test

Thus

LL-BB

frog peptic cells) but

suggested

to act

as

a

competitive antagonist to BB. Whether

these differences

relate to subtypes

of B B

receptors

remains

to be elucidated although

pancreatic and peptic cell receptors could not be

distinguished by markedly

different affinities for B B or LL-BB.

exhibit

reduced

tissues

competition

binding LL-BB

sites. to rat

competition pancreas

affinities for frog curves

However brain

curves

had

and

Hill slopes

in the presence

membranes were

rat brain

no

indistinguishable

1157

I suggesting

of guanine

longer

(data not shown).

<

receptors

did

in these

heterogeneous

nucleotides binding of

displayed from

LL-BB and

those

such

behavior

obtained

with

and rat

Vol. 157, No. 3, 1988 In summary,

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS LL-BB exhibits little or no intrinsic a c t i v i t y for a number

of BB receptors and practically studies

indicate that

cells.

The

it

functions

as an antagonist

is capable of stimulating

pseudopeptide

may

therefore

to BB.

Our

BB receptors on peptic

exhibit

differential

intrinsic

efficacy for BB receptors in d i f f e r e n t tissues.

ACKNOWLEDGMENTS The authors would like to thank Debbie Beam for manuscript preparation. This work was aided by Grant IN-66-28 from the American Cancer Society to Kenneth E.J. Dickinson and by NIH Grant # CA-45153 to David H. Coy. We also g r a t e f u l l y acknowledge support from the Gastroenterology Education and Development Fund.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

9. 10. 11. 12.

Zachary, I. and Rozengurt, E. (1985). Proc. Natl. Acad. Sci. (USA). 82, 7616-7620. Cuttitta, F., Carney, D . N . , Mulshine, J . , Moody, T.W., Fedorko, J . , Fischler, A. and Minna, J . D . (1985). Nature 316, 823-826. Jensen, R . T . , Jones, S.W., Folkers, K. and Gardner, J . D . (1984). Nature, 309, 61-63. Heinz-Erian, P., Coy, D , H . , Tamura, M., Jones, S.W., Gardner, J . D . and Jensen, R . T . (1987}. Am. J. Physiol. 252, G439-G442. Coy, D . H . , Heinz-Erian, P., Jiang, N . Y . , Sasaki, Y . , Taylor, J . , Moreau, J . P . , Wolfrey, W . T . , Gardner, J . D . and Jensen, R . T . (1988). J. Biol. Chem. 263, 5056-5060. Shirakawa, T. and Hirschowitz, B . I . Am. J. Physiol. (1985). 249, G668-G673. Sasaki, Y. and Coy, D.H. (1986). Peptides 8, 119-121. Amsterdam, A . , Solomon, T . E . and Jamieson, J . D . (1978). Methods Cell Biol. 20, 362"-378. Rick, W. and Stebauer, H.P. (1974). In Methods of Enzymatic Analysis (Bergmeyer, H.V., Ed.) pp 885-895, Academic Press, New York, NY. Matsumoto, H., Dickinson, K.E.J., Shirakawa, T., Komiyama, K. and Hirschowitz, B.I. (1987). Am. J. Physiol. 253, G557-G565. Matsumoto, H., Dickinson, K.E.J., Anderson, W. and Hirschowitz, B.I. (1988). Life Sci. 42, 1237-1244. Cowan, A., Wheeler, H. and Coy, D.H. (1988). F A S E B J. 2, A784.

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