Cholecystokinin elevates cytosolic calcium in small cell lung cancer cells

Vol. 163, No. 1, 1989 August

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages

30, 1989

CHOLECYSTOKININ

ELEVATES Julie

Department

of Biochemistry School

Received

July

25,

CYTOSOLIC CALCIUM IN SMALL CELL LUNG CANCER CELLS

Staley,

University

605-610

Gary

Fiskum

and Terry

and Molecular

of Medicine

W. Moody*

Biology,

and Health

The George

Sciences,

Washington

Washington,

D.C.

20037

1989

The ability of cholecystokinin (CCK) to elevate intracellular Ca*' levels in small cell lung cancer cells was investigated using the fluorescent Ca" indicator Fura 2. CCK-8 elevated the cytosolic Ca" levels in cell line NCI-H345 in a dose dependent manner. Nanomolar concentration of CCK-8 Ca '+ levels elevated cytosolic in the absence or presence of extracellular Ca". Potent CCK agonists such as gastrin-1 and nonsulfated CCK-8 but not inactive compounds such as CCK-27-32-NH2 elevated cytosolic Ca" levels. These data suggest that CCK receptors may regulate the release of Ca" from intracellular organelles in small cell lung cancer cells. 0 1989 Academic Press,

Cholecystokinin periphery

(1).

neurons

(2).

(CCK) is

In the brain,

CCK-B receptors

a brain

neuromodulator

neurons

of the myenteric

receptors

in

pancreatic

the

agent

certain

CNS and peripheral

cells

are (14).

(12).

synthesized Because

*TO whom correspondence Ctr., Dept. Biochemistry D.C. 20037.

with rodent

plexus

(7).

pancreatic

active

high

affinity

binding

CCK is

data

that

sites

and may function

localized

organs

as

to intrinsic to CCK-A

(8-10).

In

turnover,

In the periphery,

indicate

some CNS

CCK may bind

and other

(8,ll).

CNS and

in

phosphatidylinositol

secretion

the

dopamine

When released, acini

in

with

behavior

In the periphery,

These

may also

colocalized

may interact

CCK stimulates

satiety

biologically

CCK alters

(6).

and amylase

Peptides

it

(3-5).

stomach,

acini,

cGMP levels

peptides

CCK is

When released

termed

a peptide

~nc.

45Ca efflux, CCK is

CCK is biologically

a potent

active

in

cells. be active in

BN binds

(13)

in malignant and secreted

to cell

surface

cells. from

Bombesin small

receptors

cell (15),

(BN)-like lung

cancer

elevates

should be addressed at The George Washington Univ. h Molecular Biology, 2300 Eye St. NW, Washington,

0006-291X/89 605

SCLC

Med.

$1.50

Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

BIOCHEMICAL

Vol. 163, No. 1, 1969

cytosolic

Ca'+ (16)

autocrine

growth

identified Ca"

and stimulates

factor.

growth

Recently,

on SCLC cells

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(18).

high

Here

it may function

(17), affinity

as an

CCK-8 binding

we investigated

if

sites

CCK elevates

were cytosolic

in SCLC cells.

MATERIALS

AND METHODS

The human tumor cell line NCI-H345 was cultured in serum-supplemented medium (RPMI 1640 containing 10% heat inactivated fetal calf serum) in a humidified atmosphere of 5% CO2 and 95% air at 37'C. One day after a medium change the cells were harvested by centrifugation at 1,000 x g for 10 minutes. Cells were washed and resuspended in SIT medium (RPM1 1640 containing 3 x 10" M Na,SeO,, 5 pg/ml bovine insulin, and 10 pg/ml human transferrin (Sigma Chemical Co., St Louis, MO)). SCLC cells (2.5 x 106/ml) were resuspended in SIT medium which contained 20 mM HEPES/NaOH (pH 7.4) for loading of the fluorescent Ca2' indicator Fura 2 AM (19). Cell suspensions (10 ml) were incubated with 5 pM Fura 2 (Calbiochem Inc., La Jolla,CA) at 37°C for 30 minutes in a shaking water bath. Cells were centrifuged at 150 x g for 2 minutes, and the pellet containing SCLC cells loaded with Fura 2 was resuspended (2.5 x lo6 cells/ml). The loaded cells were transferred to a spectrofluorometer equipped with a magnetic stirring mechanism and a temperature (37'C) regulated cuvette holder. Fluorescence was monitored at excitation and emission wavelengths of 340 nm and 510 run respectively. Cytosolic Ca2+ concentrations were calculated employing methods similar to those used previously (20) with correction for the presence of extracellular Fura 2. Synthetic CCK analogues were obtained from Peninsula Laboratories, San Carlos, CA.

RESULTS The basal

cytosolic

Previously,

we found

that

Ca2+ levels

to 190 nM.

BN-like

Here

manner.

Figure

1A shows

levels.

Within

15 seconds

levels

weakly

cytosolic

the

(Fig.

lC-E).

cytosolic

investigated.

Ca2+ that added

there

peptides

0.1

after

transiently

(Fig,

1B).

increased These

data

not

10,

the

alter

the

cytosolic cytosolic

that

Ca2' Ca"

100 or 1000 nM CCK-8,

150 to 180 nM, followed

indicated

cytosolic

Ca2' in a dose dependent

of 1 nM CCK-8 the

Using

from

elevated

cytosolic

nM CCK-8 did addition

was 150 nM -+ 20 nM.

the

by a rapid

CCK-8 transiently

increases

Ca2' levels.

The source

was added

Ca2' concentration

CCK-8 elevated

that

increased

Ca2+ strongly

decline

free

of

the

CCK-induced

CCK-8 caused there

a strong

was a decrease

was bound

in the

to extracellular

was a significant

increase

increase

in

cytosolic

Ca'+ response relative Fura-2. in 606

(Fig.

cytosolic

When 5 mM EGTA

2A).

fluorescence Nonetheless,

the

Ca2+ was

due to removal when Ca2+

almost

CCK-8 was identical

of

BIOCHEMICAL

Vol. 163, No. 1, 1969

A 0.1

nM

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

B 1.0

CCK-0

nM

C ecu-a

10

E

D

nM

100

CCK-8

nM

CCK-8

1000

I

nM

I

Time

Figure 1. Dose response curve of CCK-8. The ability of (A) 0.1 nM CCK-8, (B) 1 ti CCK-8, (C) 10 nM CCK-8 (D) 100 nM CCK-8 and (E) 1000 nM CCK-8 to elevate This cytosolic Ca" in SCLC cell line NCI-H345 was determined using Fura 2. figure is representative of 3 other determinations. to

that

that

obtained

in

CCK-8 causes

elevated data

of extracellular

presence

release

The ability investigated.

the

of Ca2+ from

of other

the

indicate

as CCK receptor

cytosolic that

CCK-8,

Ca2' whereas CCK-8,

agonists

intracellular

CCK analogues

At a 10 nM dose,

to elevate gastrin-1,

A

data

cytosolic nonsulfated did

nonsulfated CCK-27-32-NH2

These

indicate

pools.

CCK-27-32-NH,

gastrin-1, whereas

Ca2'.

not

Ca2' was CCK-8 and CCK-4

(Table

I).

These

CCK-8 and CCK-4 may function is

inactive.

Peptides

B

5mM

EGTA

1 10 nM CCK-8 k-f-7

Time

Finure 2. free Ca2+. the absence concentration

Ca" response to Fura 2 measurements (A) and presence of 10 nM.

CCK-8

in were

(B)

of

the

presence and absence of extracellular in the presence of 0.4 mM CaCl, CCK-8 was added at a 5 mM EGTA. made

607

in

CCK-8

Vol. 163, No. 1, 1989 Table

BIOCHEMICAL I.

Ability

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of CCK analogues

Peptide

to elevate

cytosolic

Ca"

Ca2+ response

CCK-8

+

gastrin-1

+

nonsulfated

CCK-8

+

CCK-4

+/-

CCK-27-32-NH, The ability of various CCK agonists (10 nM) to elevate indicated. (+), Strong response; (+/-), weak response; structure of the gastrin I and CCK is shown below: Gastrin

cytosolic Ca2+ is (-) inactive. The

pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-GluGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-N& Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2

I

CCK-8

so3

unrelated

structurally

polypeptide

had no effect

CCK-transiently decline.

The decline

depletion

of

on the

increased

as somatostatin

cytosolic the

Ca"

cytosolic

Ca2+ pools.

Initially,

When 10 nM BN was subsequently

response

3A).

1 A 1OnM

Similarly, a strong

addition

10 nM BN

10

rather

than

10 nM CCK-8 caused added

(Fig.

B

CCK-8

by a rapid

there

3B).

a strong

was a strong

of 10 nM neurotensin

Ca2' response

intestinal

(16).

desensitization

Ca*+ response. (Fig.

and vasoactive

Ca2' followed

may be due to receptor

intracellular

of 10 nM CCK-8 caused

E

to CCK such

after

Ca*'

addition

In contrast,

addition

C

nM CCK-8

IO nM NT

10 nM CCK-8

10 nM CCK -8

c

U-0-J

4

IL

/ 2min

Figure 3. Ca" response in the presence measurements were made after addition of 10 nM CCK-8 followed by 10 nM neurotensin nM CCK-8 (C).

608

of various

Fura 2 peptides. 10 nM CCK-8 followed by 10 nM BN (A), (B) and 10 nM CCK-8 followed by 10

1

BIOCHEMICAL

Vol. 163, No. 1, 1989

of 10 nM CCK-8 after (Fig.

3C).

These

Ca*'

through

initial data

distinct

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

addition

indicate

of 10 nM CCK-8 resulted

that

CCK, neurotensin

in no response

and BN elevate

neuropeptide

receptors.

we characterized

receptors

for

(Kd - 2.4

nM) to a single

cytosolic

DISCUSSION Previously, '251-CCK-8

bound

with

high

CCK-8,

(Bmax = 1700/tell). inhibited

specific

CCK-33

CNS or CCK-B type.

we investigated

data.

Previously,

it

turnover

release

of Ca*+ from

Ca*+ response,

CCK-8 may cause

plasmalemmal

but

to elevate

cytosolic

growth

that

and L-365,260

cell

line

and

than

may be of the to

manner.

with

the

that

The ED,, for

IC,, obtained

from

CCK-8 stimulated

inositol-1,4,5

trisphosphate

pools.

Because

of EGTA had no effect of Ca*+ from

the

on the

intracellular

released

chelation

of

CCK-8 induced stores

and

10 nM gastrin-1

CCK-4

(10 nM) caused Preliminary

a weak Ca*' data

or 10 t-&i L-365,260

10 nM L-365,260 Ca*+ levels. were

and nonsulfated

demonstrated

response

(J.

Staley,

whereas

the

ability

CCK-

on the basal of 10 nM CCK-8

the benzodiazepine

to be potent

a

unpublished)

had no effect

antagonized Previously,

CCK-8 caused

analogues

CCK-A and CCK-B receptor

(21,22).

Besides using

The

release

10 nM L-364,718

Ca*+ levels,

antagonists

dependent

agrees

intracellular

(10 nM) was inactive.

364,718

6.5

affinity

of CCK agonists

was determined

addition

to CCK-8,

Ca*+ response.

that

4.5,

Ca*+ influx.

In addition

indicate

1.4,

higher

ability

of sites

CCK-8 and CCK-4

SCLC CCK receptors

the

closely

(11).

Ca2+ by the

27-32-NH,

class

of 0.9,

slightly

Ca2' in a dose

extracellular

strong

with

CCK-8,

1 nM which

phosphatidylinositol

not

IC,, values

CCK-8 bound

cytosolic

CCK-8 was approximately

may cause

nonsulfated

(18).

Ca*+ in SCLC cells.

CCK-8 elevated

the binding

with

or nonsulfated

Here

cytosolic

CCK-33,

binding

Because

gastrin-1,

elevate

gastrin-1,

='I-CCK-8

25 nM respectively. did

affinity

CCK in SCLC cells

CCK, BN and neurotensin NCI-H345

of SCLC in vitro

(13,23). (17,24).

release

Ca*+ from

BN and neurotensin Currently, 609

we are

intracellular stimulate

investigating

pools the if

clonal CCK

L-

BIOCHEMICAL

Vol. 163, No. 1, 1969 alters

the

growth

of SCLC cells.

CCK elevates

the

biologically

active.

cytosolic

Ca"

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

In summary, indicates

that

the demonstration

that

SCLC CCK-B receptors

are

ACKNOWLEDGMENTS This 48071.

research

The authors

is

supported

thank

Drs.

by NCI-grants R. Jensen

CA-32946,

and S. Mahmoud

33767, for

42306

and

helpful

discussions. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

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