Platelet-derived growth factor activation of gastric mucosal calcium channels

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1496-1502

Vo1.186, No. 3,1992 August 14,1992

PLATELET-DERIVED

GROWTH MUCOSAL

FACTOR

ACTIVATION

CALCIUM

OF

GASTRIC

CHANNELS

B.L. Slomiany, J. Liu,

and A. Slomiany

Research Center, New Jersey Dental School University of Medicine and Dentistry of New Jersey Newark, NJ 07103 Received June 29, 1992

- Gastric mucosal calcium channel complex was isolated from the s o l u b i l i z e d e p i t h e l i a l cell m e m b r a n e s by a f f i n i t y chromatography on wheat germ agglutinin. The complex following labeling with [3H]PN200-100 was reconstituted into phospholipid vesicles which exhibited active 45Ca2+ uptake. The channels responded in a dose dependent manner to dihydropyridine calcium antagonist, PN200-110. which at 0.5~M exerted maximal i n h i ~ i t o ~ affect of 66% on 45Ca 2+ uptake, while a 52% enhancement in ~ C a ~uptake occurred with a specific calcium channel activator, BAY K8644. On platelet-derived growth factor (PDGF) binding in the presence of ATP, channels showed an increase in protein tyrosine phosphorylation of 55 and 170kDa subunits of calcium channel. Such p h o s p h o r y l a t e d c h a n n e l s f o l l o w i n g r e c o n s t i t u t i o n into vesicles displayed a 78% greater 45Ca2+ uptake. The results point towards the importance of PDGF in the regulation of gastric mucosal calcium homeostasis. ©1992AcademicPress, Inc. SUMMARY

INTRODUCTION

-

The

depends

a

delicate

upon

preservation balance

of

gastric

mucosal

of

factors

which

processes of mucosal repair and restitution. is

the

maintenance

of

intracellular

integrity

control

the

Primary among these

calcium

levels.

This

important regulatory element is recognized for its participation in

many

cellular

differentiation shown

to

involving Hence,

secretion

cells

occurs

activity

(1,2),

calcium through

voltage

capable

including

cellular

conditions,

specific

factors

channels

and

potentiate

physiological secretory

processes,

of

contraction,

and

its

influx

injury

(3).

entry

in most

carefully the

been

normal

excitatory

co~trolled

and

process

channels

expression

could be of significance

has

Under

and receptor-dependent affecting

cell

of

(1).

calcium

to the processes

of

mucosal integrity maintenance and repair. Primary among these is a potent mitogen, platelet-derived growth factor (PDGF). The

biological

effects

of

PDGF

located on the target cell surfaces 0006-291X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

1496

are

(4-6).

mediated

by

receptors

The ligand binding of

Vol. 186, No. 3, 1992

the

receptor

"BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

results

tyrosine

kinase

tyrosine

residues

event

towards

in

activation

activity

the

and phosphorylation

(4-6).

mediating

of

This the

intrinsic of the

autophosphorylation

proliferative

receptor

receptor

on

is a central

effects

of

PDGF.

In

this report, we describe the isolation of calcium channel complex from

rat

stimulated uptake

gastric channel

into

mucosal protein

phospholipid

cell

membranes

phosphorylation vesicles

and

show

affects

containing

that the

PDGFcalcium

reconstituted

calcium channels. AND METHODS F r e s h l y d i s s e c t e d rat s t o m a c h s w e r e opened along the greater curvature, rinsed with ice-cold 0.10M NaCl in 0.05M phosphate buffer pH 7.2, and the mucosal cells were collected by scraping the mucosa with a blunt spatula. Scrapings were placed in ice-cold buffer (2.5mM Tris-HCl, pH 7.0, 250mM sucrose, 2.5mM EDTA, imM phenyl methyl sulfonyl fluoride (PMSF), i00 TIU/ml aprotinin and l#g/ml leupeptin, and homogenized for 1 min in a polytron tissuemizer. The homogenate was centrifuged at 400xg for 15 min at 4oC and NaCl/MgSO 4 added to the supernatant to form final concentrations of 0.1mM and 0.2mM, respectively (7). After centrifugation of the supernatant at 40,000xg for lh at 4°C, the pellet was resuspended in 0.1mM sodium phosphate buffer, pH 7.2 and s t o r e d at -70°C u n t i l use. Protein concentration of the resuspended pellet was estimated using the BCA protein assay kit. The m e m b r a n e p r e p a r a t i o n was c e n t r i f u g e d for 20 m i n at 1 0 , 0 0 0 x g at 4°C, and the p e l l e t s o l u b i l i z e d u s i n g a b u f f e r containing 50mM T r i s - H C l , pH 7.4, 0 . 5 M NaCl, 20mM 3-[(3cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS), 10% glycerol, I00 TIU/ml aprotinin, l#g/ml leupeptin, an imM PMSF. After lh at 4°C, the mixture was centrifuged at 105,q00xg for 60 min and the resulting supernatant collected, inM of [~H]PN200-110 was added and incubated at 4°C for 30 min. The preparation was then applied to a ~olumn of Sepharose-bound wheat germ agglutinin, and the [~H]PN200-110 labeled channel protein was eluted with buffer containing 300mM N-acetylglucosamine (8). P D G F b i n d i n g a s s a y s w e r e c a r r i e d out ~ i n c u b a t i n g the membrane samples (300~g protein/assay) with [ ~ I ] P D G F (0.016nM) and 5mM Tris/HCl (pH 7.0), 125mM sucrose, 75mM NaCl, 0.5mM CaCI2, 0.5% bovine serum albumin (BSA) in a final volume of 200~I (7). Unlabeled PDGF was added to the incubations to form a final concentration of 0.66nM in the experiments where nonspecific binding was being estimated. Incubates were maintained for lh at room temperature, and then stopped by addition of ImM ice-cold buffer containing 10mM Tris/HCl (pH 7.0) and 0.5% B S ~ . Membranebound [125I]PDGF was separated from the unbound [ ~ 5 I ] P D G F by centrifugation at 10,000xg for I0 min at 4°C. The effect of dihydropyridine calcium antagonist, PN200-110, on the binding of PDGF was assessed following preincubation of membrane preparation with PN200-110 (0-200~M) at room temperature for 30 min. For PDGF-stimulated channel protein phosphorylation, the solubilized calcium channel preparations containing 200-300~g protein were incubated with 50mM HEPES buffer, pH 7.6, 10mM MgSO4, ImM PMSF and with or without 1.4nM PDGF, or with 0.1mM Ophospho-l-tyrosine. After 30 min at 25°C, phosphorylation was initiated by the addition of a solution containing 10#M ATP, imM CTP, 8mM MnCI2, 20mM MgCI2, and 2mM s o d i u m v a n a d a t e (7)° Incubations were maintained for i0 min at 4°C after which the MATERIALS

1497

Vol. 186, No. 3, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

calcium channel preparation was recovered using wheat germ agglutinin affinity chromatography. For SDS-PAGE, the reaction mixture was treated with 170mM Tris-HCl buffer, pH 6.8, containing 10% SDS and 100mM dithiothreitol, an heated at 100°C for 5 min. Following electrophoresis, the proteins were transferred onto 0.2~m nitrocellulose membranes. Ten p e r c e n t BSA was u s e d to b l o c k the m e m b r a n e s , mouse anti-phosphotyrosine monoclonal IgG (10~g/ml), as detecting antibody, and goat antimouse alkaline phosphatase conjugated IgG (0.1~g/ml) as secondary antibody (9). Liposomes were prepared by the method of Mimms et al. (I0). Purified calcium channel samples in t h e i r i n t a c t f o r m and following P D G F - s t i m u l a t e d p h o s p h o r y l a t i o n were solubilized with a buffer containing 20mM CHAPS, 5mM CaCI2, 50mM Tris-HCl, pH 7.4, and 10% glycerol, and mixed with iml of o c t y l g l u c o s i d e containing 1% egg y o l k p h o s p h a t i d y l c h o l i n e . Detergent was removed by dialysis against buffered saline, thus yielding liposomes. For further purification, a suspension of liposomes was made with 45% sucrose, overlaid with 2ml of 30% sucrose and iml of 10% sucrose and then centrifuged at 4°C for 18h at 45,000xg in a Beckman SWS0 r o t o r (ii). The liposomes containing [3H]PN200-110 labeled channel protein were recovered as a white band at the top of 10% sucrose layer. For the m e a s u r e m e n t s of 4 5 C a 2 + uptake into vesicles containing the reconstituted calcium channels, external divalent cations were removed by column chromatography on Sephadex G-50 (12). U p t a k e into v e s i c l e s (i00~i) in s u c r o s e medium was initiated by additioD of 50~i of sucrose m e d i u m c o n t a i n i n g 0.1mM CaCl2 plus 2~Ci of 45Ca2+. The mixture was incubated at 37°C for varlous periods of time up to lh, and the reaction was t e r m i n a t e d with 150mM MgCI 2 in 10mM HEPES-Tris buffer, pH 7.4. The effect of calcium channel receptor antagonist, PN200-110 and that of calcium channel activator, BAY K8644 on the 45Ca2+ uptake was measured following vesicles p r e i n c u b a t i o n at room temperature for 20 min with PN200-110 (0-1~M) or BAY K8644 (010~M). The samples were then applied to a Chelex i00 column (0.5 x 5cm) equilibrated with sucrose medium c o n t a i n i n g img/ml BSA. Vesicles were eluted from the column with iml of sucrose-BSA medium. Gastric

R E S U L T S

-

gastric

epithelium

channels.

The

a and

Sepharose-bound with

medium

which

Upon

PDGF

tyrosine region

wheat

germ

to

the

was

of

130,

110,

with

receptor the

column

yielded On

on the

SDS-PAGE

gave five major p r o t e i n 90

an

observed

rat

calcium

chromatography

complex.

the'preparation 170,

of

prelabeled

Elution

protein

from

channel

affinity

phosphorylation,

phosphorylation

were

calcium

N-acetylglucosamine

channel at

prepared

isolation

agglutinino

containing

condition,

migrated induced

of

and

55kDa

increase the

(Fig. in

proteins

1) o

protein in

the

of 170 and 55kDa.

Figure mucosal

for

membranes

subjected

labeled

under reducing

used

membranes

dihydropyridine

buffer

[3H]PN200-100 bands

were

cell

solubilized

[3H]PN200-110, antagonist,

mucosal

2

shows

receptor

the

binding

effect of PDGF. 1498

of

PN200-110

The binding

on

the

gastric

assays gave a mean

Vol. 186, No. 3, 1 9 9 2

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

c

V

kDa 170-

-7

i o

116-

v

1

=

865848-

o ....

.11111111

////////2

~///////. ,,I,,

,///Z///I

////I

,//H//.,..

o. ZlI////.'I

.///Z////

n

;5555555; iiiiiiii.

......

----

9///////,

111/111..

0

5O

o

@

1

2

- - / I I I I I I I /

. . . . . . . .

@

100

150

PN200-1 IO(~M)

Fig. I. Effect of PDGF on gastric mucosal calcium channel proteins tyrosine phosphorylation (line 2). The purified channel preparation was incubated with PDGF (l.4nM) for 30 min and then ATP substrate (10~M) was added, and the reaction stopped after i0 min. Following SDS-PAGE, the proteins were transferred onto nitrocellulose membranes and probed with mouse antiphosphotyrosine monoclonal IgG. Visualization was achieved using alkaline phosphatase conjugated goat anti-mouse IgG and 5-bromo4-chloro-3-indole phosphate/nitro blue tetrazolium. Line i, channel protein pattern under reducing conditions visualized by silver stain. Fig, 2. Effect of PN200-110 on the PDGF binding to gastric mucosal calcium channel protein. Assays were conducted as described in Materials and Methods, except that membranes prior to binding assay were preincubated at room temperature for 30 min with 0-200~M PN200-110. Values represent the means ± SD of five separate experiments performed in duplicate.

specific value of 1.46 fmol/mg m e m b r a n e p r o t e i n PN200-110,

and

preincubation

was

with

not

significantly

calcium

channel

in the absence of

different

receptor

following

antagonist,

PN200-

ii0. The

function of the isolated

following

incorporation

phosphatidylcholine membrane

bound

reconstituted exhibited to

that

of

protein.

gastric

reconstituted

calcium

channel

protein

which

conform

the

calcium

45Ca2+ uptake

protein-free

dependent manner

channels

Under

mucosal

6-fold greater of

the

vesicles

calcium

vesicles.

channels

to PN200-110,

the

assay

activator.

The maximal

inhibitory 1499

complex

channel

into

structure

protein

of the

complex

into v e s i c l e s

as c o m p a r e d

The

containing

responded

as well as to BAY K8644,

evaluated

conditions,

vesicles in

a

a dihydropyridine

antagonist,

was

a specific effect

was

concentration calcium

calcium attained

channel channel at

0.5~M

Vol. 186, No. 3, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1~"6

= x

-:2 Io

i

I

I

0

0,1

0.2

I

J

I

0.3

0,4

0.8

2

'

0

PN200-110(~M,A)

®

i

i

i

i

i

0

2

4

6

8

A Bay

B

C

D

K8644(~M,O)

3. Effect of PN200-110 ~ ia. DCa z~ into vesicles containing

and BAY K8644 on the uptake of the reconstituted gastric mucosal calcium channels. Uptake assays were conducted as described in Materials and Methods using vesicles preincubated for 20 min at room temperature with different concentrations of PN200-110 (00.8~M) or BAY K8644 (0-8~M). Values represent the means ± SD of five separate experiment performed in duplicate. Fi~. ~. Effect of PDGF-stimulated calcium channel protein phosphorylation on 45Ca2+ uptake into vesicles containing the reconstituted gastric mucosal calcium channels. The purified channel protein was reconstituted into phospholipid vesicles either in its intact form (C) or following PDGF-induced phosphorylation (D). A and B, vesicles free of membrane protein in the absence (A) and the presence (B) of PDGF. Values represent the means ± SD of five experimetns performed in duplicate. PN200-110 45Ca2+

at which

uptake

enhancement To

concentration

occurred,

of a n t a g o n i s t BAY

K8644

in 45Ca2+ uptake at 6~M

ascertain

whether

represents

transport

binding

the

to

while

into

vesicular

the

calcium

The

concentration thus

data

(0.7mM/ml)

indicating

intravesicular The

is

shown

45Ca2+

vesicles

at

the

vesicles

and

not

45Ca2+-preloaded

11%

at

vesicles

its

45Ca2+

of c a l c i u m

mere

from the v e s i c l e

lanthanum

only

the m a j o r i t y

uptake

into

purified

in Fig.

was

maximum

that

by

space

the c a l c i u m

that

caused

in

(52%)

was

optimal

displacement, present

in the

space and hence i n a c c e s s i b l e to La 3+ displacement.

45Ca2+

phosphorylated

revealed

uptake active

the

were t r e a t e d with La 3+ to d i s p l a c e surface°

maximal

(Fig. 3).

osmotically surface,

a 66% d e c r e a s e

caused

4.

In

proportional 30 min.

containing

vesicles

gastric both to

However,

containing

mucosal types

the

time

the

PDGF-induced

of of

extent

the

calcium

channel

vesicles,

the

incubation of u p t a k e

phosphorylated

and

protein

uptake

and of

calcium

was 78% g r e a t e r as c o m p a r e d to that of the controls. 1500

intact

of

reached

45Ca2+

by

channels

Vol. 186, No. 3, 1992

DISCUSSION

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PDGF is a potent mitogen capable of stimulation of

-

epithelial

cell p r o l i f e r a t i v e

While

majority

the

considered

to

be

of a

and s e c r e t o r y r e s p o n s e s

proliferative

direct

effects

consequence

of

of

(4-6).

PDGF

are

phospholipase

C

activation for polyphosphoinositide hydrolysis and the release of calcium from intracellular stores, that

the

earliest

factor

receptors

recent

patch

suggest

responses

involve

clamp

that

to

the

the

of

of

data the

are

obtained

EGF

presented

calcium

channels

that

herein

in gastric

phosphorylation

activation

provide

of PDGF

on

receptor

to

the

growth Indeedg

A431

cells

triggers

the

The results of

for

cell

tyrosine leads

with

(14).

evidence

indications

(13).

receptor

epithelial

the

of

calcium

receptor-operated calcium channel response studies

strong

activation

influx

recording

activation

there

the

presence

membrane,

residues stimulation

of

and

show

through

the

of

calcium

channel activity. The solubilized calcium channels, labeled with [3H]PN200-110 and purified by affinity chromatography on wheat germ agglutinin, following

incorporation

into

the

phosphatidylcholine

exhibited active 45Ca2+ uptake and responded dependent

manner

antagonist, activator. mucosal using in

PN200-110,

a

dihydropyridine

calcium

as well as to BAY K8644, a specific calcium channel On PDGF binding in the presence of ATP,the gastric

calcium

tyrosine

to

vesicles

in a concentration

channels

responded

phosphorylation.

anti-phosphotyrosine

phosphorylation

was

by

an

Examination

of

antibody

increase the

protein

revealed that

reflected

mainly

in

in

this

55

and

protein patterns increase 170kDa

proteins.

The vesicles containing phosphorylated channel protein

displayed

a 78%

tyrosine

kinase

activation.

greater

calcium

involvement

in

uptake,

PDGF

thus

dependent

calcium

the

channel

This finding together with the data from patch clamp

recording at the level of single ion channels the

indicating

expression

of calcium channel

(14) suggest that

activity depends

not only

on

the phosphorylation events controlled by protein kinase A and C (15,16),

but

also

on

the

phosphorylation

triggered

by

PDGF

receptor activation. The fact that the activation of calcium channels in gastric mucosa

is

dependent

towards

the

calcium

influx,

on

importance an

the of

event

PDGF

this of

receptor

mitogen

significance

mucosal repair and integrity maintenance. 1501

in to

activation the the

points

regulation

of

processes

of

Vol. 186, No. 3, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACKNOWLEDGMENTS - This w o r k was s u p p o r t e d by U S P H S G r a n t DK21684-15 from the National Institute of Diabetes and Digestive and Kidney Diseases, and Grant AA05858-II from the National Institute on Alcohol Abuse and Alcoholism, NIHo

REFERENCES

i. 2. 3. 4. 5. 6. 7. 8. 9,

i0. Ii. 12. 13. 14. 15. 16.

Hosey, M.M,, Lazdunski, M. (1988) J. Membr. Biol. 104,81105. Kass, G,E,N., Llopis, J., Chow, S.C., Duddy, S.K., Orrenius, S. ( 1 9 9 0 ) J. Biol. Chem. 265, 17486-17492. Smith, M.H., Thor, H., Orrenius, S. (1981) science 213, 1257-1259. Carpenter, G. (1987) Ann. Rev. Biochem. 56, 881-914. Decker, S.J., and Harris, P. (1989) J. Biol. Chem. 264, 9204-9209. Kelly, J.D., Haldeman, B.A., Grant, F.J., Murray, M.J., Seifert, R.A., Rowen-Pope, D.F.,Cooper, J.A., and Kazlaukas, S. (1991) J. Biol. Chem. 266, 8987-8992. Slomiany, B.L., Liu, J., Yao, P., Wu-Wang, C.Y., Keoghe J.P., Wang, S.L., and Slomiany, A. (1990) Digestion 47, 181-190. Jay, S.D., Sharp, A.H., Kahl, S.D., Vedvick, T.S., Harpold, M.M., and Campbell, K.P. (1991) J. Biol. Chem. 266, 32873293. Bielanski, W., Keogh, J.P., Wang, S.L., Liu, J., Konturek, S.J., Slomiany, A., and Slomiany, B.L. (1991) Biochem. Int. 25, 419-427. Mimms, L.T., Zampighi, G., Nozaki, Y., Tanford, C., and Reynolds, J.A. (1981) Biochemistry 20, 833-840. Sengupta, S., Shovlin, F.E., Slomiany, A., and Slomiany, B.L. (1991) Int. J. Biochem. 23, 115-121. Curtis, B.M., and Catterall, W.A. (1986) Biochemistry 25, 3077-3083. Pandiella, A., Magni. M,, Lovisolo, D., and Meldolesi J. (1989) J. Biol. Chem. 264, 12914-12921. Peppelenbosch, M.P., Tertoolen, L.G.F., deLaat, S.W. (1991) J. Biol. Chem. 266, 19938-19944. Nunoki, K., Florio, V., Catterall, W.A. (1989) Proc. Natl° Acad. Sci. USA 86, 6816-6820. O'Callahan, C.M., Ptasienski, J., Hosey, M.M. (1988) J Biol Chem. 263, 17342-17349.

1502