Positive inotropic effects and receptors of calcitonin gene-related peptide(CGRP) in porcine ventricular muscles

Vo1.155, No. 1, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 289-294

August 30, 1988

POSITIVE INOTROPIC EFFECTS AND RECEPTORS OF CALCITONIN GENE-RELATED PEPTIDE(CGRP) IN PORCINE VENTRICULAR MUSCLES Miyauchi,

T.,

Department *Department **Tsukuba

**Sano, Y., **Hiroshima, 0., *Ishikawa, T., *,***Saito,

**Yuzuriha,

T.,

Sugisbita,

Y.,

A. and *Goto, K.

of Internal Medicine, Institute of Clinical Medicine and of Pharmacology, Institute of Basic Medical Sciences, University of Tsukuba, and Research Laboratories, E i s a i Co. L t d . , T s u k u b a , I b a r a k l 305 / a p a r t

Received June 15, 1988

Summary: C a l c i t o n i n gene-related peptide (CGRP), b u t n o t c a p s a i c l n , elicited positive inotropic effects in the isolated, electrically driven false tendon of the porcine heart. Specific CGRP-binding sites were present in solubilized membrane f r a c t i o n s ; the dissociation constant (Kd) a n d t h e m a x i m u m b i n d i n g (Bmax) w e r e 5 0 . 4 pM a n d 180 f m o l / m g p r o t e i n , respectively. SDS-PAGE a n a l y s i s of CGRP-binding sites revealed the molecular m a s s o f 7 0 K a n d 1 2 0 K. Few CGRP-like immunoreactive nerves were present in the ventricular muscle layer. T~ese results indicate t h a t CGRP a c t i v a t e s specific receptor sites on the ventricular muscles and causes positive inotropic responses. CGRP r e c e p t o r s in ventricles are likely to be activated by circulating CGRP. © 1988 Ao~aem±c Press, Inc.

Immunohistochemical calcitonin

gene-related

not only

in the nervous

positive

inotropic

guinea

pig

atria

conflicting exerted pigs

positive

pigs

cord

pharmacological tendon potential

or

and is

(1,2) an

of the

inotropic

have

a 37 a m i n o a c i d p e p t i d e , but

also

increase

likely nerves

in plasma

in

to

the

function

as

of

effects

in the strips

that

CGRP e l i c i t s tension,

However

of

have

muscles.

perfused

and papillary

there

hearts

muscles

a

in the

a neurotransmitter

CGRP o n v e n t r i c u l a r isolated,

shown

is distributed

(3-5).

contractile

(6,8,9).

effect

in the ventriculaz

studies

been CGRP

of guinea of rats

and

(7,11).

Recently spinal

system

noncholinergic results

(10) but not

guinea

radioimmunologlcal

peptide(CGRP),

effect, (6,7)

nonadrenergic,

and

specific (:[2)

and

effects

and ventl~icular role

binding human

sites

placenta

and binding muscles

of

o f CGRP i n c o n t r o l l i n g

*** To whom c o r r e s p o n d e n c e

for

CGRP were (13).

characteristics porcine

hearts,

the cardiac

isolated In

the

from the present

porcine study,

were examined in the respectively,

to

false

assess

a

function.

should be addressed.

Abbreviations: CGRP: c a l c i t o n i n gene-~elated dodecyl sulfate-polyacrylamide gel electrophoresis.

289

peptidc;

SDS-PAGE:

sodium

0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 155, No. 1, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

M e t h o d s and M a t e r i a l s F r e s h p o r c i n e h e a r t s were o b t a i n e d from a local slaughter house and ventricles of hearts were isolated i n an i c e - c o l d K r e b s - R i n g e r W s s o l u t i o n . The c o m p o s i t i o n o f t h e K r e b s - R i n g e r ' s s o l u t i o n was NaC1, 113; KC1, 4 . 8 ; CaC1 z , 2 . 2 ; KH2PO4, 1 . 2 ; MgSO4, 1 . 2 ; NaHC03, 25; and g l u c o s e , 5 . 5 (mM). Pharmacological studies in vitro: Since thick ventricular walls are difficult to maintain in a sufficient condition to analyze the pharmacological effects, t h i n f a l s e t e n d o n s ( a b o u t 2 mm t h i c k , 10 mm l o n g ) w e r e i s o l a t e d and s u s p e n d e d in organ baths filled with a Krebs-Ringer's solution ( 3 7 oC) c o n s t a n t l y b u b b l e d w i t h 95 O2 and 5 CO2. The i s o m e t r i c c o n t r a c t i o n was m e a s u r e d b y a t r a n s d u c e r and r e c o r d e d on a t h e r m a l p e n r e c o r d e r . The t i s s u e was applied w i t h a r e s t i n g t e n s i o n o f 1 g and d r i v e n e l e c t r i c a l l y a t 1 Hz w i t h s q u a r e wave pulses (1 m s e c , 2 V) g e n e r a t e d by a stimulator. When t h e t w i t c h contractions a t t a i n e d a s t e a d y s t a t e , human CGRP o r i s o p r o t e r e n o l was a d d e d i n a cumulative manner. After washing followed by a resting period of 1 hr, c a p s a i c i n was a p p l i e d . Binding studies: Ventricles were minced and homogenized with a buffer containing 10 mM T r i s - H C 1 (pH = 7 . 4 ) , 0 . 5 mM p h e n y l m e t h y l s u l f o n y l f l u o r i d e , 1 mM e t h y l e n e d i a m i n e t e t r a a c e t a t e , and 1 ~ g / m l e a c h o f a n t i p a i n , leupeptin, p e p s t a t i n A and p h o s p h o r a m i d o n . The h o m o g e n a t e s were c e n t r i f u g e d a t 1500 X g f o r 10 m i n , and t h e s u p e r n a t a n t was c e n t r i f u g e d a t 5 0 , 0 0 0 X g f o r 30 m i n . Then the pellets were resuspended i n t h e same b u f f e r (crude membrane fraction). The c r u d e m e m b r a n e f r a c t i o n s were treated w i t h 3% d i g i t o n i n , s o n i c a t e d and c e n t r i f u g e d a t 1 0 0 , 0 0 0 X g a t 4 oc f o r 40 m i n . The s u p e r n a t a n t s were designated as solubilized fractions. The p r o t e i n concentration was d e t e r m i n e d b y t h e m e t h o d o f Lowry e t a l . (14) u s i n g b o v i n e s e r u m a l b u m i n a s the standard. The b i n d i n g a s s a y o f CGRP w a s c a r r i e d out according to the method described previously by Hiroshima et al. (12). In brief, t h e c r u d e membrane and s o l u b i l i z e d fractions w e r e m i x e d w i t h [ l ~ S I ] h u m a n a-CGRP (18 t o 23 pM). A f t e r i n c u b a t i o n a t 0 °C f o r 15 h r s , b o v i n e y - g l o b u l i n and p o l y e t h y l e n e g l y c o l 6000 w e r e a d d e d . The f r e e and b o u n d r a d i o l i g a n d s w e r e t h e n s e p a r a t e d b y r a p i d filtration. The r a d i o a c t i v i t y in the filters was c o u n t e d u s i n g A l o k a ARC-300 y-counter. The s p e c i f i c b i n d i n g was d e f i n e d a s t h e d i f f e r e n c e o f t h e amount of bound radioactivity b e t w e e n t h e a b s e n c e and p r e s e n c e o f u n l a b e l e d human a CGRP ( 1 . 1 ~M), For the analysis of the molecular weight, the solubilized f r a c t i o n was i n c u b a t e d w i t h [ x z S I ] h u m a n a-CGRP ( 0 . 1 1 nM) a t 0 oc e i t h e r i n t h e p r e s e n c e o r a b s e n c e o f 3 . 0 ~M u n l a b e l e d human a-CGRP. The [ x ~ S I ] h u m a n a - C G R P - b i n d i n g s i t e c o m p l e x was t h e n s e p a r a t e d f r o m f r e e l i g a n d b y g e l f i l t r a t i o n and incubated with disuccinimldyl suberate. Covalently labeled binding sites were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) b y t h e m e t h o d o f Laemmli ( 1 5 ) . Standard molecular markers were myosin heavy chains (Mr = 200 K ) , c a l d e s m o n (Mr = 155 K ) , a - a c t i n i n (Mr = 105 K ) , d e s m i n (Mr = 55 K ) , a - t r o p o m y o s i n (Mr = 43 K ) , a c t i n (Mr = 42 K) and ~ - t r o p o m y o s i n (Mr = 35 K ) . After electrophoresis, t h e g e l was e x p o s e d t o X - r a y f i l m f o r 2 w e e k s . Immunohistochemistry: The f a l s e t e n d o n s and v e n t r i c u l a r walls were fixed in p a r a f o r m a l d e h y d e and p i c r i c a c i d f o r 72 h r s and p r o c e s s e d f o r a f l u o r e s c e n c e i m m u n o h i s t o c h e m i s t r y a c c o r d i n g t o t h e m e t h o d d e s c r i b e d b y S a i t o and Goto ( 2 ) .

Results

In ventricles, manner

the

isolated,

electrically

CGRP i n d u c e d p o s i t i v e

(1 nM t o 300 n M ) ( F i g .

produced dose-dependent

1).

positive

driven

inotropic

false responses

Isoproterenol, inotropic 290

tendon in

of

porcine

a dose-dependent

a ~-adrenergic

responses

the

in a similar

agonist, dose

also range.

Vol. 155, No. 1, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

CGRP(-log M)

o.3,1

2 4 6 B (pM)

-

1C 5rain

capsaicin 5

C

20

h

40

60

Totaladded (pe)

/

100-

B (~M)

Q

(9

10

20

30

Total added (pM)

-log M

Figure

1. T y p i c a l e x a m p l e o f t h e e f f e c t o f CGRP (A) and d o s e - r e s p o n s e c u r v e s for the positive i n o t r o p i c e f f e c t o f CGRP and i s o p r o t e r e n o l (B) on t h e contraction of ventricular muscles. Numbers i n t h e f i g u r e i n d i c a t e t h e - l o g M. Each p o i n t and b a r r e p r e s e n t s t h e mean and S . E . o f 7 t i s s u e s . The ED50 v a l u e s ( w i t h 95% c o n f i d e n c e i n t e r v a l ) t o CGRP and i s o p r o t e r e n o l i n t h e s e p r e p a r a t i o n s were 5 . 5 0 nM ( 1 . 6 4 - 1 . 8 8 nM) and 3 6 . 9 nM ( 1 2 . 7 107 nM), r e s p e c t i v e l y . The maximum r e s p o n s e i n d u c e d b y CGRP was 3 3 . 9 ± 3.2% o f t h a t b y i s o p r o t e r e n o l .

Figure

2. Total, nonspecific and specific binding of [12sI]CGRP and Scatchard analysis t o t h e c r u d e m e m b r a n e (A) a n d s o l u b i t i z e d (B) fractions of ventricular muscles. Each value represents t h e mean o f four duplicate determinations.

However,

the

Capsaicin,

maximum response which

positive

releases

inotropic

caused

a slight

membrane

analysis

fmol/mg

and

revealed

an equilibrium

solubilized

inhibited solubilized ~- and rat

in

the

in

fractions a-CGRP, but

all

the

contractile

2 are

the

saturation

of

a

constant

crude

were not

0.85

of

of

porcine saturable

fractions (n = 4). ICS0 values 22.0 II

291

nM,

(0.48

isoproterenol.

(9)

elicited

of

[lzsI]human

but

7 tissues.

a-CGRP t o

the

Scatchard

binding

component

pM a n d a d e n s i t y and 50.4

no

(n = 7), the

ventricles.

8.7

of

to

examined

in 5 out

single,

nM a n d

angiotensin

preparations

curves

respectively

that

nerves

(Kd)

membrane

human a-CGRP;

30% o f

tension

fractions

presence

about

intracardiac

the

fractions,

by unlabeled

from

of

solubilized the

CGRP w a s

CGRP

dissociation

protein

to

response

decrease

Shown in Figure

the

+i°4o

r.... - ~ , / J : " - ~ Isoproterenol -.o-- CGRP

0

crude

B o.s~--'---

(Bmax)

with of

35

pM a n d 1 8 0 f m o l / m g

in

[125I]human-CGRP-binding

was

in

and

the

crude

respectively pM) n o r

membrane (Fig.

3).

human calcitonin

Human (0.15

V o l . 155, N o . 1, 1 9 8 8

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

ventricle h CGRP

(-)

(+)

Mr ~:-200K (- 155K (- I05K

10(1

('- 55K

~- 43K ~- 35K

11

10

9

8

7

I

6

UnLabeledhumano~-CGRP(-logM)

F i g u r e 3. Inhibition o f [ x 2 s I ] C G R P - b i n d i n g t o t h e c r u d e membrane ( o p e n circle) and s o l u b i l i z e d ( c l o s e d c i r c l e ) f r a c t i o n s of the v e n t r i c u l a r m u s c l e s by u n l a b e l e d human a-CGRP. Each p o i n t r e p r e s e n t s t h e mean o f duplicate determinations. F i g u r e 4. D e t e r m i n a t i o n o f t h e m o l e c u l a r s i z e o f CGRP-binding s i t e s by c r o s s - l i n k i n g of [x2sI]CGRP f o l l o w e d by SDS-PAGE. The numbers r e p r e s e n t t h e m o l e c u l a r w e i g h t o f marker p r o t e i n s .

pM), not

inhibited

the

binding

of

[I~sI]CGRP

to

the

solubillzed

fractions

(data

shown). SDS-PAGE

intense

analysis

radioactive

bands were

specific

Few muscle

the

at

70 K a n d 120 K ( F i g .

t o CGRP s i n c e

CGRP-like

layers.

present

bands

of

CGRP-binding

it

around coronary

beaded

blood

fibers

The

the

were

observed

in

fibers

a s shown p r e v i o u s l y

presence

labeling

by unlabeled

varicose-type

vessels

revealed

4).

was p r e v e n t e d

immunoreactive

In contrast,

sites

of

of

these

CGRP. the

were

ventricular found

to

be

(16).

Discussion

CGRP a n d

isoproterenol

produced

positive

inotropic

similar dose range, although the maximum response that by

isoproterenol.

positive

inotropic

(7).

In

guinea

isolated,

Previous

effects

pigs,

perfused

studies

in ventrioular

CGRP

hearts

strips

augmented

(i0) but not

heart

rate.

response

the

contractile Thus

was a c t u a l l y

it

is

force not

the

contraction

in the

isolated,

by affecting

certain

induced by

what

infused 292

was

in

smaller

that CGRP did not

and p a p i l l a r y

papillary muscles and ventricular strips (II). may c h a n g e

by CGRP

indicated

responses

muscles

than induce

of rats

of ventricles

the

perfusion

CGRP.

of

The

the

in

electrically driven

In perfused preparations,

extent

a

pressure positive

CGRP-induced

CGRP

and/or inotropic positive

Vol. 155, No. 1, 1988

inotropic

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

responses

in

were unequivocally Recently

specific

1 . 2 5 pmol/mg p r o t e i n of

CGRP t o

in

fairly

the

those

at

sites

analysis

cyclase

G-protein

of

to

fractions

The b i n d i n g

it

However,

solubilized

distinct

(13).

CGRP s i n c e

human placenta.

that

was

the

inhibited

Kd a n d Bmax

of ventricles

This

indicates

f r o m and h a v e

CGRP-binding

Recently

receptors

it

are

coupled

If

is possible

it

is

were

that

higher

the

affinity

characteristics

intense

shown that

CGRP a c t i v a t e s

muscles

the

(7,11).

It

is

systems

are

t h e CGRP r e c e p t o r

is coupled

with

the

components with

complex

possible

another

radioactive

adenylate-cyclase

that

also

gave

and atrial to

(17).

( 1 2 0 K) r e p r e s e n t s

molecular

was

(16)

sites

a receptor-G-protein

Alternatively

weight

it

arteries

with G-protelns

weight

respectively.

the

1 2 0 K.

(Mr = c a 40 K ) ,

low m o l e c u l a r

exact

specific

are

in coronary

associated

molecular

muscles

in the placenta.

documented

usually

ventricular

human p l a c e n t a

nor calcitonin.

and

in the

in ventricles

70 K and

adenylate well

those

driven

study.

f o r CGRP w i t h Kd a n d Bmax o f 1 . 8 7 nM a n d

from the

II

membrane

than

SDS-PAGE bands

sites

isolated

m e m b r a n e s was

crude

smaller

electrically

in the present

not by anglotensin

CGRP-binding than

isolated,

binding

were

ventricular

b y CGRPs b u t values

the

demonstrated

that

single

and

the

and

component with high

molecule.

of CGRP-binding sites

a high

CGRP r e c e p t o r ,

Admittedly

in the ventricles

the

remain

to be determined. Stimulation

of

chronotroplc

and

It

been

has

also

noncholinerglc present

in

observation

shown

nerves the

(21)

inotropic

responses

positive

indicates

and

a poor

muscles,

in

supply

although

in

the

plasma

patients also

the

levels

positive causes

in humans

with medullary

has

been of

of

of

circulating

Like

guinea

CGRP,

pigs

and

capsaicin

(9,10).

the

acts (20),

Therefore,

in

the

but

porcine

scarcely previous o n CGRP-

causes

muscles

(18,19).

coronary positive

the not

lack

of

o f CGRP,

ventricular

are present.

studies

h a v e shown t h e p r e s e n c e

rats

thyroid

with

deferentia

on ventricular nerves

were

Capsaicin

CGRP i n v a s a

positive

pigs

nonadrenergic,

fibers

in agreement (16).

elicits

guinea

in these

immunoreactlve

CGRP-contalning

(3-5).

The p l a s m a

carcinoma

that

pentagastrin

(23).

in isolated 293

of CGRP-like material levels

and in pregnant

Intravenous

in humans

responses

(3)

calcium,

CGRP ( 4 , 5 ) .

responses

inotropic

a transmitter

walls

capsaicin of

atria

ventricles,

(9,22).

demonstrated

inotropic positive

CGRP i s

CGRP r e c e p t o r s

Radioimmunological

nerves of

and releases

atria of

nonchollnergic isolated

CGRP-like

hearts

effect

in

ventricular

in vitro

arteries

any

that

layer

left

nerves

effects

(6,8).

muscle

in the

containing

nonadrenergic,

inotropic

o f CGRP r i s e

in

humans

It

or capsaicin

infusion

of

Coronary perfusion hearts

of

guinea

(4).

elevates

CGRP i n d u c e s o f CGRP a l s o pigs

(10).

Vol. 155, No. 1, 1988

These

findings

circulating

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

suggest

that

ventricular

CGRP-receptors

are

activated

by

CGRP.

Acknowledgments:

We t h a n k L . G .

manuscript.

work was s u p p o r t e d

This

Project Research,

Bond f o r

advice

in preparation

of

the

b y g r a n t s from U n i v e r s i t y of l s u k u b a

the N a i t o F o u n d a t i o n , 7apan F o u n d a t i o n f o r H e a l t h S c i e n c e ,

Kowa L i f e S c i e n c e F o u n d a t i o n ,

the

Research

Foundation

for

Pharmaceutical

S c i e n c e s and G r a n t - i n - A i d from the M i n i s t r y of E d u c a t i o n , S c i e n c e and C u l t u r e of Yapan.

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