Vol. 173, No. 1, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 252-258

November 30,1990

ADRENAL PHEOCHROMOCYTOMR PCI2H CELLS RESPOND TO PITUITARY ADENYLATE CYCLASE ACTIVATING POLYPEPTIDE

Takuya W a t a n a b e ~ Tetsuya Ohtaki, Chieko Kitada, M a s a h i k o Fujino

Masao Tsuda and

Tsukuba R e s e a r c h Laboratories, Takeda Chemical Industries, Wadai 7, Tsukuba, Ibaraki 300-42, Japan

Ltd.,

Received October 12, 1990 An adrenal p h e o c h r o m o c y t o m a cell line, PCI2h, was found to respond to a novel h y p o t h a l a m i c neuropeptide, P i t u i t a r y Adenylate Cyclase A c t i v a t i n g P o l y p e p t i d e (PACAP). The cells e l e v a t e d b o t h i n t r a c e l l u l a r and e x t r a c e l l u l a r c A M P l e v e l s on s t i m u l a t i o n by PACAP, w h e r e a s t h e y s h o w e d l i t t l e r e s p o n s e to V I P w h i c h is s t r u c t u r a l l y r e l a t e d to PACAP. U s i n g [125I]PACAP27 (a s h o r t e r form of the p e p t i d e ) and [125I]VIP, we f o u n d l a r g e a m o u n t s of specific b i n d i n g sites for PACAP but few b i n d i n g sites for VIP in PCI2h cells. These results indicate that PCI2h cells respond to PACAP via a specific PACAP receptor. ©1990AcademicPress,Inc. SUMMARY:

The

PC12

cell

line

is d e r i v e d

adrenal pheochromocytoma

(i).

et al.

that VIP

clase

(3) h a v e activity

reported of P C 1 2

adenylate cyclase t i o n of V I P real

cells.

required

Tischler

a transplantable

et al.

stimulates

However,

the

modulator

This

the

for the P C 1 2

adenylate

activation

observation

rat

(2) and R o s k o s k i

an u n p h y s i o l o g i c a l l y

(i pM or more).

physiological

from

of

cythe

high concentrasuggests

cells

exists

that and

a it

might be a VIP related peptide rather than VIP itself. Recently, ly related

M i y a t a et al. isolated

to VIP

two novel p e p t i d e s close-

from ovine h y p o t h a l a m i c

tissues,

named

Pitui-

To whom correspondence should be addressed. A b b r e v i a t i o n s : PACAP38, p i t u i t a r y a d e n y l a t e c y c l a s e a c t i v a t i n g p o l y p e p t i d e with 38 residues and amidated C-terminus; PACAP27, a s h o r t e r f o r m p e p t i d e w i t h 27 r e s i d u e s c o r r e s p o n d i n g to the Nterminal 27 amino acids of PACAP38 and amidated C-terminus; VIP, v a s o a c t i v e i n t e s t i n a l p o l y p e p t i d e ; HBSS, Hanks' b a l a n c e d salt solution; HEPES, N - 2 - H y d r o x y e t h y l p i p e r a z i n e - N ' - 2 - e t h a n e s u l f o n i c acid; CHAPS, 3 - [ ( 3 - C h o l a m i d o p r o p y l ) d i m e t h y l a m m o n i o ] - l - p r o p a n e sulfonate; BSA, bovine serum albumin. ooo6-291x/9o $1.5o Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

252

Vol. 173, No. 1, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

tary Adenylate

Cyclase

(PACAP38)

and

(4),

of PACAP38 to t h a t

are r e v e a l e d

is

activity tration PCI2h

(5).

(68%

homology

study was

a physiological of PC12

cells.

of PACAP38

cells

concentration

of V I P

the response.

Moreover,

binding

but,

cells. function

sites, Based of

on

PACAP

a

the N - t e r m i n u s

27 r e s i d u e s

structures

with

modulator

and rats to test for

stimulated

of PC12

cells

required

are h i g h l y

N-terminus

undertaken

the that

evidence,

(7)),

VIP

we

peripheral

residues)

cyclase

a nanomolar

concen-

the

cyclase

the such

of

a micromolar

same

extent

of PACAP

binding

tissue,

that

adenylate

whereas

discuss

and

(6).

the a d e n y l a t e

to o b t a i n

few

28

homologous

a hypothesis

we found large amounts

in contrast,

in

38 r e s i d u e s

We d e m o n s t r a t e

was

this

with

in humans

or PACAP27

(a subclone

Polypeptide,

one w i t h

Their

to be c o n s e r v e d

The p r e s e n t PACAP

the other

(PACAP27)

of V I P

Activating

sites

of

specific in PCI2h

physiological as

the

adrenal

glands.

MATERIALS

AND

METHODS

M a % e r l a l s : P C l 2 h c e l l s (7) w e r e k i n d l y p r o v i d e d by Dr. H a t a n a k a (Institute for P r o t e i n Research, Osaka University). N e w b o r n calf s e r u m w a s p u r c h a s e d from M i t s u b i s h i C h e m i c a l I n d u s t r i e s (Tokyo, Japan), cAMP assay kits from A m e r s h a m (Buckinghamshire, England), [125I]VIP ( 2 2 0 0 C i / m m o l ) f r o m N e w E n g l a n d N u c l e a r ( B o s t o n , MA), and h u m a n VIP from the Peptide Institute (Osaka, Japan). Peptide synthesis and radioiodina%ion: P A C A P 3 8 and P A C A P 2 7 was synthesized by an automatic synthesizer (ABI model 430A). PACAP27 was iodinated by the peroxidase m e t h o d as d e s c r i b e d p r e v i o u s l y (8). C y c l i c AMP assay: PCI2h cells were g r o w n in D u l b e c c o ' s m o d i f i e d Eagle's medium supplemented w i t h 10% n e w b o r n c a l f s e r u m in a h u m i d i f i e d a t m o s p h e r e of 95% a i r a n d 5% CO 2 at 37°C. For the c A M P assay, c e l l s w e r e p l a t e d on c o l l a g e n - c o a t e d 4 8 - w e l l m u l t i well p l a t e s at a b o u t 5 x 1 0 4 c e l l s / w e l l . The c e l l s w e r e u s e d at 7 to i0 days after plating. The m e d i u m was r e p l a c e d w i t h 500 ~i of H B S S i n c l u d i n g 0 . 0 5 % BSA. Following incubation at 37°C for 30 min, P A C A P or V I P w a s c h a l l e n g e d to t h e c e l l s . After the i n c u b a t i o n at 37°C, t h e c e l l s w e r e w a s h e d t w i c e w i t h H B S S and t h e n i n t r a c e l l u l a r c A M P was e x t r a c t e d w i t h 20% p e r c h l o r i c acid. The e x t r a c t was n e u t r a l i z e d w i t h 1.5M KOH c o n t a i n i n g 6 0 m M H E P E S and s u b j e c t e d to r a d i o i m m u n o a s s a y . E x t r a c e l l u l a r C A M P was assayed by a p p l y i n g aliquots of the i n c u b a t e d m e d i u m directly. Binding assay: C e l l s in 2 4 - w e l l m u l t i w e l l p l a t e s w e r e w a s h e d w i t h H B S S i n c l u d i n g 0 . 0 5 % B S A and 0 . 0 5 % C H A P S ( b i n d i n g b u f f e r ) and incubated at 37°C f o r 1 h r i n t h e p r e s e n c e of 30 pM [I~SI]PACAP or [125I]VIP in 200 ~i of the b i n d i n g buffer. A f t e r the

253

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B I O C H E M I C A L A N D B I O P H Y S I C A L RESEARCH C O M M U N I C A T I O N S

completion of the binding, the cells b u f f e r a n d t h e n s o l u b i l i z e d w i t h 500 quots were counted by a gamma-counter.

were washed N1 of 0.5 M

with NaOH

the and

same ali-

RESULTS Exposure elevation at

of

8 min),

control) within

of

an

PCI2h

intracellular

reaching

by

25

60 m i n

its

min

(Fig.

at

10 -6 M o f

of P A C A P 3 8 PACAP38

was

(Fig.

1000-fold

PACAP.

As

creased

and

level the

PCI2h in

Fig.

1 0 -7 M

The of

times

that

was

to

of

the

baseline

of i n t r a c e l l u l a r

minimum

VIP

control

that

reduce

PACAP27

secreted 3,

a plateau

at

not

of

a rapid

of t h e c o n t r o l effective

was

the

very

dose

same

weak

as

(about

cAMP production.

of

cAMP

by

extracellular

level

dose-dependently

level

the

increase

PACAP,

cells

that

i00 t i m e s

potency to

in

(20

did

2).

in s t i m u l a t i n g

reached

increased

The

Compared

indicated

control

and reached

10 -I° M.

Furthermore,

level

sustained

(Fig.

resulted

(i0 t i m e s

level

This

PACAP38

weaker)

PACAP38

cAMP

the

I).

2).

to

maximal

and

cAMP was dose-dependent level

cells

and

by

120

reached

PACAP38

the

addition

of

cAMP

slowly

in-

min.

This

200

(Fig.

times

4).

plateau that

As

in

of the

20i o-~

03

10

0

0

o

8

\

6

%

n

o_o/ /

%

°~~o

o.O

E 10

/o/

4

13.. < 0

oo