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E F F E C T S O F I S O P R O T E R E N O L AMD F O R S K O L I N O N T E N S I O N , C Y C L I C AMP L E V E L S A N D C Y C L I C A M P - D E P E N D E N T PROTEIN KINASE ACTIVITY IN BOVINE CORONARY

ARTERY

by

VENKATA KRISHNAM RAJU

VEGESNA

B. P h a r m . , A n d h r a U n i v e r s i t y , M. P h a r m . , A n d h r a U n i v e r s i t y ,

A THESIS THE

India, India,

1978 1980

SUBMITTED I N P A R T I A L FULFILMENT REQUIREMENTS FOR M A S T E R OF

THE

DEGREE

OF

OF

SCIENCE

in THE

FACULTY

OF

GRADUATE

STUDIES

D i v i s i o n o f P h a r m a c o l o g y and T o x i c o l o g y of the F a c u l t y of P h a r m a c e u t i c a l S c i e n c e s

We

THE ©

a c c e p t t h i s t h e s i s as conforming to the r e q u i r e d s t a n d a r d

U N I V E R S I T Y OF B R I T I S H C O L U M B I A O c t o b e r 1983

V e n k a t a K r i s h n a m R a j u V e g e s n a , 1983

In

presenting

requirements of

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f o r an advanced

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Columbia, I agree that available

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The U n i v e r s i t y o f B r i t i s h 1956 Main M a l l Vancouver, Canada V6T 1Y3

Date

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(3/81)

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Columbia

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ACKNOWLEDGEMENTS

I would l i k e t o e x p r e s s my h e a r t f e l t g r a t i t u d e to my s u p e r v i s o r Dr. Jack Diamond, Ph.D.

for his excellent

guidance,

encouragement, p a t i e n c e and s u p p o r t i n c o m p l e t i n g t h i s work. I would a l s o l i k e t o e x p r e s s my s i n c e r e thanks t o Mrs. E v e l y n Chu f o r her c o o p e r a t i o n and h e l p d u r i n g the c o u r s e o f t h i s work. L a s t but not l e a s t , I am g r a t e f u l

to a l l my c o l l e a g u e s

and f r i e n d s f o r t h e i r encouraging c r i t i c i s m , v a l u a b l e d i s c u s s i o n s , and t i m e l y h e l p which c o n t r i b u t e d to a g r e a t e x t e n t i n c o m p l e t i n g t h i s work.

Raju V.K.

Vegesna

-i i-

ABSTRACT The e f f e c t s of isoproterenol and f o r s k o l i n on t e n s i o n , c y c l i c AMP (cAMP) l e v e l s , and c y c l i c AMP-dependent protein kinase (cA kinase) a c t i v i t y were compared in h e l i c a l s t r i p s of bovine coronary a r t e r y . Isoproterenol and f o r s k o l i n produced time-dependent and dose-dependent increases in cAMP l e v e l s and both compounds relaxed potassium-contracted a r t e r i e s .

Relaxation and cAMP elevation appeared

to be well correlated i n the isoproterenol experiments.

However, i n

contrast to the r e s u l t s with i s o p r o t e r e n o l , cAMP elevation and r e l a x a t i o n were not well correlated at lower concentrations of f o r s k o l i n . For example, 0.1 uM f o r s k o l i n increased cAMP l e v e l s i n the a r t e r i e s by approximately 5.5 f o l d but did not relax the muscles.

A smaller

elevation of cAMP produced by 1.0 uM i s o p r o t e r e n o l , on the other hand, was accompanied by an almost complete r e l a x a t i o n of a r t e r i e s . Physiological processes which are thought to be mediated by cAMP are assumed to be a consequence of s e l e c t i v e a c t i v a t i o n of c y c l i c AMP-dependent protein kinase (cA kinase).

It i s possible that

low doses of f o r s k o l i n may elevate c y c l i c AMP in a compartment which does not have access to these kinases, whereas, higher doses may

—i i i —

i n c r e a s e cAMP l e v e l s i n a l l compartments. t h i s p o s s i b i l i t y , optimal conditions

In o r d e r t o i n v e s t i g a t e

were e s t a b l i s h e d

i n our

laboratory

f o r t h e measurement o f cA k i n a s e a c t i v i t y i n c o r o n a r y

arteries.

I t has been suggested t h a t a d d i t i o n

buffer

o f 0.5 M NaCl t o t h e

used f o r homogenization o f c o r o n a r y a r t e r i e s i s e s s e n t i a l i n

o r d e r t o demonstrate hormonal e f f e c t s on cA k i n a s e i n t h i s t i s s u e . However, under o u r e x p e r i m e n t a l c o n d i t i o n s , strength-'in

t h e homogenization b u f f e r

cA k i n a s e a c t i v i t y .

the i o n i c

significantly inhibited total

I n t e r e s t i n g l y , i n agreement w i t h p r e v i o u s r e p o r t s

i n o t h e r t i s s u e s , an apparent t r a n s l o c a t i o n from t h e c y t o s o l

increasing

o f cA k i n a s e a c t i v i t y

t o t h e p a r t i c u l a t e f r a c t i o n was observed under con-

d i t i o n s a s s o c i a t e d w i t h high t i s s u e l e v e l s o f cAMP. r e l a x a t i o n caused by i s o p r o t e r e n o l

For example,

was accompanied by i n c r e a s e d cA

k i n a s e a c t i v i t y i n t h e p a r t i c u l a t e f r a c t i o n and decreased a c t i v i t y i n the s o l u b l e

f r a c t i o n o f the coronary a r t e r i e s .

A s i m i l a r s h i f t i n the

d i s t r i b u t i o n o f t h e k i n a s e was caused by v a r i o u s c o n c e n t r a t i o n s o f f o r s k o l i n , i r r e s p e c t i v e o f whether t h e a r t e r i e s were r e l a x e d

or not.

Thus, as was observed w i t h cAMP l e v e l s , a c t i v a t i o n o f cA k i n a s e d i d not always c o r r e l a t e w e l l w i t h r e l a x a t i o n

i n this tissue.

Other workers have suggested t h a t low doses o f f o r s k o l i n can potentiate

hormonally-induced e f f e c t s i n several

suggested t h a t such a p o t e n t i a t i o n

tissues.

would c o n s t i t u t e

f a v o u r o f a r o l e f o r cAMP i n t h e hormonal response.

I t was

evidence i n However, i n t h e

p r e s e n t e x p e r i m e n t s i n c o r o n a r y a r t e r i e s , i t was found t h a t low doses o f f o r s k o l i n , which s i g n i f i c a n t l y i n c r e a s e d cAMP l e v e l s , d i d not potentiate

isoproterenol-induced relaxation or elevation

o f cAMP.

-IV-

Only h i g h e r doses o f f o r s k o l i n (>_ 1 yM) p o t e n t i a t e d i s o p r o t e r e n o l induced cAMP g e n e r a t i o n .

These r e s u l t s suggest t h a t c a u t i o n

be e x e r c i s e d i n u t i l i z i n g f o r s k o l i n as a t o o l

should

i n elucidating a role

o f c y c l i c n u c l e o t i d e s i n v a s c u l a r smooth muscle f u n c t i o n . Our

r e s u l t s suggest t h a t cAMP may n o t be t h e o n l y mechanism

r e s p o n s i b l e f o r r e l a x a t i o n o f c o r o n a r y a r t e r i e s by these drugs o r a l t e r n a t i v e l y , t h a t some form o f f u n c t i o n a l

compartmentalization

o f cAMP and cA k i n a s e e x i s t s i n t h i s t i s s u e .

Jack Diamond Supervisor

- V -

CONTENTS

Page ABSTRACT

i i

LIST OF TABLES

vi i i

LIST OF FIGURES

Ix

INTRODUCTION:

A.

C y c l i c AMP : General View

1

B.

Role o f cAMP i n t h e c o n t r o l o f Smooth Muscle C o n t r a c t i o n

3

C.

C y c l i c AMP-dependent P r o t e i n Kinase (cA K i n a s e )

9

D.

F o r s k o l i n and i t s Role i n C y c l i c N u c l e o t i d e RGSG clinch

•••••

•••••

SPECIFIC GOALS OF THE PRESENT INVESTIVATION

11

15

MATERIALS AND METHODS A.

Materials

B.

Methods: I. II. III.

16

P r e p a r a t i o n o f muscle samples

17

Measurement o f c y c l i c AMP

17

P r e p a r a t i o n o f e x t r a c t s and a s s a y o f c y c l i c AMP-dependent p r o t e i n k i n a s e

18

-vi-

Page IV. V.

Protein determination

19

Statistical

20

analyses

RESULTS A.

C o n d i t i o n s f o r t h e Assay o f C y c l i c AMP-dependent P r o t e i n Kinase (cA K i n a s e ) i n I n t a c t T i s s u e

B.

Time Course S t u d i e s o f I s o p r o t e r e n o l

21

and

F o r s k o l i n on C y c l i c AMP l e v e l s and T e n s i o n i n B o v i n e Coronary A r t e r i e s C.

23

E f f e c t s o f Various Concentrations o f Isoproterenol

and F o r s k o l i n on cAMP L e v e l s and

T e n s i o n i n Coronary A r t e r i e s D.

E f f e c t s o f F o r s k o l i n and

24

Isoproterenol,

A l o n e and i n C o m b i n a t i o n , on C y c l i c AMP L e v e l s and T e n s i o n i n Bovine Coronary A r t e r i e s . . E.

A c t i v a t i o n o f C y c l i c AMP-dependent

25

Protein

Kinase i n B o v i n e Coronary A r t e r i e s by I s o proterenol F.

and F o r s k o l i n

26

E f f e c t o f NaCl on S o l u b l e P r o t e i n

Kinase'

Activity G.

27

Effect of Addition

o f C y c l i c AMP t o t h e Homo-

genate on S u p e r n a t a n t and P a r t i c u l a t e

Protein

Kinase A c t i v i t y H.

28

Effects o f Isoproterenol Protein culate

and F o r s k o l i n on

Kinase A c t i v i t y i n Supernatant Fractions

ship to Relaxation

and'Parti-

o f _ t h e Coronary A r t e r i e s : . R e l a t i o n o f t h e Coronary A r t e r i e s

29

Page

DISCUSSION

64

SUMMARY AND CONCLUSIONS

74

REFERENCES •••••

•••*•

•••••

76

-viii-

LIST OF TABLES Page

I.

E f f e c t s o f V a r i o u s Agents on C y c l i c AMP L e v e l s and T e n s i o n i n Blood V e s s e l s

II.

5

E f f e c t s o f I s o p r o t e r e n o l and F o r s k o l i n on C y c l i c AMP L e v e l s and T e n s i o n i n Bovine Coronary A r t e r y

III.

45

E f f e c t s o f I s o p r o t e r e n o l and F o r s k o l i n on S o l u b l e C y c l i c AMP-dependent P r o t e i n Kinase A c t i v i t y Measured i n t h e Presence and Absence o f 0.5M NaCl

IV.

54

E f f e c t s o f I s o p r o t e r e n o l and F o r s k o l i n on C y c l i c AMP L e v e l s , S o l u b l e and P a r t i c u l a t e C y c l i c AMP-dependent P r o t e i n K i n a s e A c t i v i t y and T e n s i o n i n Bovine Coronary AK*16V*y

•••••

•••>•

•••••

•••••

63

-ix-

LIST OF FIGURES Page 1.

Role of Cyclic AMP and Cyclic AMP-dependent Protein Kinase in Hormonal Stimulation and Physiological Response

2.

2

Effect of Incubation Time on Cyclic AMPdependent Protein Kinase (cA Kinase) A c t i vity in Coronary Arteries

3.

32

Effect of Enzyme Concentration on cA Kinase Activity in Coronary Arteries

4.

34

Effect of Magnesium Chloride on cA Kinase Activity in Coronary Arteries

5.

36

Effect of Sodium Fluoride on cA Kinase Activity in Coronary Arteries

6.

38

Effect of Triton X-100 Concentration on Particulate cA Kinase Activity in Coronary AY^tG^I GS ••»••

7.

•••••

•••••

40

Time Course for Cyclic AMP (cAMP) Elevation and Relaxation of Coronary Arteries by 1 yM Isoproterenol

8.

42

Time Course for cAMP Elevation and Relaxation of Coronary Arteries by 10 yM Forskolin

44

-x-

Page 9.

E x p e r i m e n t a l P r o t o c o l Showing A c t i o n s I n t e r a c t i o n s o f Low doses o f

and

Isoproterenol

and F o r s k o l i n on Coronary A r t e r i e s 10.

Experimental Protocol

47

Showing A c t i o n s

I n t e r a c t i o n s o f High Doses o f

and

Isoproterenol

and F o r s k o l i n on Coronary A r t e r i e s 11.

E f f e c t s o f F o r s k o l i n on induced R e l a x a t i o n

49

Isoproterenol-

and C y c l i c AMP

Elevation

i n Bovine Coronary A r t e r i e s 12.

E f f e c t of Isoproterenol induced R e l a x a t i o n

51

on F o r s k o l i n -

and C y c l i c AMP

Elevation

i n Bovine Coronary A r t e r i e s 13.

E f f e c t o f NaCl

53

i n the Homogenizing B u f f e r

cA K i n a s e A c t i v i t y i n the Supernatant Prepared from C o n t r o l 14.

on

Fractions

Coronary A r t e r i e s

E f f e c t o f NaCl i n the homogenizing on cA K i n a s e A c t i v i t y i n the

56

Buffer

Supernatant

F r a c t i o n s Prepared from A r t e r i e s T r e a t e d w i t h 1 uM I s o p r o t e r e n o l 15.

58

I n h i b i t i o n of Soluble P r o t e i n Kinase A c t i v i t y by A d d i t i o n o f N a C l - i n d u c e d Enzyme I n h i b i t i o n by D i a l y s i s

16.

60

E f f e c t o f A d d i t i o n o f cAMP to the whole Homogenate on P r o t e i n K i n a s e A c t i v i t y i n Supern a t a n t and P a r t i c u l a t e F r a c t i o n s

62

-xi -

P o s s i b l e Mechanism o f A c t i o n o f p r o t e r e n o l and tG X* 16 S

F o r s k o l i n on

Iso-

Coronary •••••

-1-

INTRODUCTION A.

CYCLIC AMP

: General

C y c l i c AMP 1957

was

View

d i s c o v e r e d by S u t h e r l a n d and h i s c o l l e a g u e s i n

as a heat s t a b l e m e d i a t o r

lysis.

The

o f e p i n e p h r i n e ' s e f f e c t s on

research demonstrating

glycogeno-

the concept o f c y c l i c AMP

(cAMP)

as a "second messenger" ( R o b i s o n , Butcher and S u t h e r l a n d , 1971) opened a p e r i o d o f remarkable p r o g r e s s

i n the u n d e r s t a n d i n g

mechanisms whereby many d r u g s , n e u r o t r a n s m i t t e r s and

has

of

hormones produce

some o f t h e i r w e l l known e f f e c t s on t a r g e t t i s s u e . F i g u r e I i l l u s t r a t e s c u r r e n t concepts cAMP as a m e d i a t o r

r e g a r d i n g the r o l e o f

o f hormonal a c t i o n i n p h y s i o l o g i c a l r e s p o n s e s .

The

hormone b i n d s t o s p e c i f i c r e c e p t o r s on the e x t e r n a l s u r f a c e o f the plasma membrane o f the c e l l adenylate cyclase. ATP

to cAMP.

The

r e s u l t i n g i n a c t i v a t i o n o f the enzyme

This a c t i v a t i o n leads to increased conversion i n c r e a s e d c o n c e n t r a t i o n o f cAMP i n the c e l l

of

then

l e a d s to a c t i v a t i o n o f the enzyme c y c l i c AMP-dependent p r o t e i n k i n a s e . P h o s p h o r y l a t i o n o f s p e c i f i c p r o t e i n s by the a c t i v a t e d k i n a s e i s b e l i e v e d t o be r e s p o n s i b l e f o r the observed C y c l i c AMP

i n the c e l l

i s hydrolyzed

p h y s i o l o g i c a l response.

i n t o 5'-AMP, a r e a c t i o n

c a t a l y z e d by one o r more c y c l i c n u c l e o t i d e p h o s p h o d i e s t e r a s e s e x i s t i n a l l mammalian c e l l s (Robison

e t al_, 1971).

that

-2-

HORMONE

PLASMA MEMBRANE | ADENYLATE CYCLASE CYTOSOL

ATP

PHOSPHODIESTERASE 5 * - AMP INACTIVE PROTEIN KINASE IP I f t CAMP R

H

ACTIVE PROTEIN KINASE

[lj [E5 +

C A M P

PHOSPHORYLATES SPECIFIC PROTEINS

PHYSIOLOGICAL RESPONSE Figure 1:

A s c h e m a t i c diagram o f t h e mechanism by which hormones and drugs a r e thought t o produce p h y s i o l o g i c a l responses i n d i f f e r e n t t i s s u e s by e l e v a t i n g c y c l i c AMP and a c t i v a t i n g c y c l i c AMP-dependent p r o t e i n k i n a s e .

R, r e g u l a t o r y

subunit

o f c y c l i c AMP-dependent p r o t e i n k i n a s e ; C, c a t a l y t i c subu n i t o f c y c l i c AMP-dependent p r o t e i n

kinase.

-3-

For t h e past twenty y e a r s , r e g u l a t i o n o f c e l l u l a r

responses

by c y c l i c n u c l e o t i d e s has been a s u b j e c t o f i n t e n s e i n v e s t i g a t i o n . This r e s u l t e d i n an e x p o n e n t i a l

i n c r e a s e i n the number o f p u b l i c a -

t i o n s i n the c y c l i c nucleotide research area.

C y c l i c AMP has been

suggested t o be a m e d i a t o r o f many hormonal f u n c t i o n s such as g l u c a gon

s t i m u l a t i o n o f hepatic glycogenolysis, l i p o l y s i s , several

processess

involved i n the c o n t r a c t i o n - r e l a x a t i o n cycle o f the heart,

s t e r o i d o g e n s i s , t h y r o g l o b u l i n s e c r e t i o n , and s a l i v a r y amylase s e c r e t i o n ( f o r d e t a i l e d r e f e r e n c e s , see Kebabian and Nathanson (eds.,) 1982).

Although

cAMP i s thought t o r e g u l a t e many c e l l u l a r

events,

i t has proven d i f f i c u l t t o f i r m l y e s t a b l i s h t h e r e l a t i o n s h i p o f cAMP l e v e l s t o p h y s i o l o g i c a l f u n c t i o n i n i n t a c t c e l l s , t i s s u e s and organisms.

Smooth muscle appears t o be one o f t h e most c o n t r o v e r s i a l

areas i n t h i s

B.

regard.

Role o f cAMP i n t h e C o n t r o l o f Smooth Muscle C o n t r a c t i o n The

precise r o l e o f c y c l i c nucleotides i n the r e g u l a t i o n o f

smooth muscle c o n t r a c t i o n has been a s u b j e c t o f c o n t r o v e r s y f o r the l a s t two decades.

A s u b s t a n t i a l amount o f data has accumulated i n

the l i t e r a t u r e e x p l o r i n g t h e r o l e o f cAMP i n t h e r e g u l a t i o n o f smooth muscle t e n s i o n and t o review a l l t h e i n f o r m a t i o n i s beyond the scope of t h i s t h e s i s .

However, t h i s t o p i c has been e x c e l l e n t l y

by s e v e r a l a u t h o r s

reviewed

p r e s e n t i n g e v i d e n c e f o r and a g a i n s t a r o l e f o r

c y c l i c n u c l e o t i d e s i n smooth muscle f u n c t i o n (Namm and Leader, 1976; Diamond, 1978; Namm, 1980; Hardman, 1981; and Kukovetz e t a l _ , 1981). Sutherland

and R a i l

(1960) f i r s t suggested t h a t

epinephrine-induced

-4r e l a x a t i o n o f smooth muscle might be r e l a t e d t o an e l e v a t i o n o f cAMP levels i n that tissue.

From t h a t time onwards, many:researchers have

attempted t o t e s t t h e general d i f f e r e n t smooth m u s c l e s .

h y p o t h e s i s o f cAMP mediated r e l a x a t i o n o f

There i s a s u b s t a n t i a l amount o f e v i d e n c e

s u p p o r t i n g t h e concept t h a t t h e r e i s a c a u s a l r e l a t i o n s h i p between i n c r e a s e s i n cAMP l e v e l s produced by g - a d r e n e r g i c

s t i m u l a n t s and o t h e r

substances and t h e i r r e l a x a n t e f f e c t s on smooth muscle.

However t h e r e

i s a l s o evidence t h a t i s i n c o n s i s t e n t with t h i s concept. S t u d i e s done by M a r s h a l l and Kroeger (1973) and Honeyman e t a l (1978) have demonstrated a temporal and q u a n t i t a t i v e c o r r e l a t i o n between the e l e v a t i o n o f cAMP l e v e l s and t h e r e l a x i n g e f f e c t o f i s o p r o t e r e n o l and o t h e r d r u g s .

E x t e n s i v e e v i d e n c e f o r a m e d i a t o r r o l e o f cAMP i n

smooth muscle r e l a x a t i o n produced by 3 - a d r e n e r g i c

drugs and o t h e r

s t i m u l a n t s o f a d e n y l a t e c y c l a s e a c t i v i t y was a l s o p r o v i d e d by Kukovetz and

his colleagues

coronary

( s e e Kukovetz e t a l _ , 1981 for

review).

In i s o l a t e d

a r t e r i e s , i s o p r o t e r e n o l , p r o s t a c y c l i n and adenosine have been

shown t o produce c o n c e n t r a t i o n dependent i n c r e a s e s i n cAMP i n c l o s e association with i t s relaxant e f f e c t s .

Kukovetz e t a l _ have shown t h a t

the e x t e n t o f changes i n cAMP i s c o n s i s t e n t w i t h a m e d i a t o r r o l e i n t h e r e l a x a n t response.

Many r e s e a r c h e r s have s t u d i e d t h e a b i l i t y o f v a r i o u s

agents t o e l e v a t e cAMP l e v e l s and t o a l t e r t e n s i o n i n s e v e r a l smooth m u s c l e s , as shown i n T a b l e 1.

The m a j o r i t y o f t h e s t u d i e s : s u p p o r t a

r o l e o f cAMP i n t h e r e l a x a t i o n o f smooth muscle.

I t should be noted

t h a t many o f these s t u d i e s l a c k time-dependent and c o r r e l a t i o n s i n demonstrating and Hardman, 1980).

dose-dependent

t h e c a u s e - e f f e c t r e l a t i o n s h i p s (Kramer

-5-

T a b l e I . E f f e c t s o f v a r i o u s agents on c y c l i c AMP l e v e l s and tension i n blood

vessles.

Animal and V e

A|*nt

Rat Aorta

Mechanical Effect

Cyclic A M P

Angiotensin Angiotensin + theophylline Cyclopropane Iaoflurane Halothane Angiotensin Angiotensin + theophylline Mepivacaine

Contraction Not reported Contraction Relaxation Relaxation Contraction Contraction Relaxation

No change Decreased Decreased Increased Increased Decreased Decreased Increased

Serotonin Nitroglycerin D-600 Dipyridamole Histamine Histamine + metiamide Histamine + mepyr amine Diazoxide MnCI, EGTA

Contraction Relaxation Relaxation Relaxation Contraction Increased contraction Decreased contraction Relaxation Relaxation Relaxation

No change Increased No change Increased Increased No change Increased No change No change No change

Carbachol Nitroglycerin

PGEj Acetylcholine

No change Relaxation Relaxation No change Relaxation Contraction Relaxation Contraction Relaxation Contraction

No change No change Increased No change Increased No change Increased No change Increased No change

Mesenteric artery

Diazoxide Hydralazine Histamine

Relaxation Relaxation Contraction

Facial artery

KC1 KCl + 0-methyldigoxin PGF,. PGEj

Relaxation Contraction Contraction Relaxation

Increased Increased Decreased at 15 s, increased at 5 min Increased Increased No change Increased

Histamine

Contraction

Increased

Tail artery Portal vein Rabbit Pulmonary artery

Mesenteric artery Mesenteric vein Dog Femoral artery Mesenteric artery Lobar artery Lobar vein Saphenous vein Cow

Common dorsal digital vein Pig

Coronary artery

Human Umbilical artery

PGEi PGFz. PGEi PGF2. PGEi

PGFJ.

Acetylcholine

Contraction Contraction Bradykinin Contraction Serotonin Contraction KCl Contraction PGEi Relaxation PGA, Contraction PGF,. Contraction Ionophore A-23187 Contraction Oxygen Contraction EGTA, ethylene glycol bis(0-aminoethyl ether) tetraacetk acid; PG, prostaglandin. Histamine

Kramer and Hardman (1980)

No change No change No change No change No change Increased No change No change No change No change

-6-

In d i r e c t c o n t r a s t t o t h e above f i n d i n g s , i t has been demonstrated i n some smooth muscles t h a t r e l a x a t i o n does n o t always o c c u r when cAMP l e v e l s are increased.

For example, i n r a t myometrium Diamond and

Holmes (1975) observed t h a t KC.l-induced d e p o l a r i z a t i o n i n c r e a s e d cAMP levels.

Instead o f r e l a x a t i o n , t h e s e e l e v a t e d cAMP l e v e l s were

accompanied by c o n t r a c t i o n o f t h e r a t myometrium.

They f u r t h e r observed

t h a t p a p a v e r i n e and n i t r o g l y c e r i n e r e l a x e d t h e s e d e p o l a r i z e d w i t h o u t any d e t e c t a b l e change i n cAMP l e v e l s . suggested t h a t i n c r e a s e s

These a u t h o r s

i n cAMP l e v e l s might n o t be r e s p o n s i b l e f o r

relaxation i n that preparation.

Supporting

these observations

and M c N e i l l

(1976) have shown a dose r e l a t e d r e l a x a t i o n o f

depolarized

r a t uteri

with isoproterenol.

f i n d i n g s Harbon and A s s o c i a t e s

proterenol

Verma

However, no a l t e r a t i o n i n

cAMP l e v e l s was observed a t any dose t e s t e d .

found i n c r e a s e s

muscles

S i m i l a r t o these

( s e e Harbon e t a l _ , 1978 f o r r e v i e w )

i n cAMP l e v e l s i n r a t myometrium t r e a t e d w i t h

o r PGE-j.

iso-

However, i s o p r o t e r e n o l produced r e l a x a t i o n where-

as PGE-j produced c o n t r a c t i o n i n t h i s t i s s u e .

These a u t h o r s q u e s t i o n e d

the e x c l u s i v e r o l e o f cAMP i n t h e r e l a x a t i o n o f smooth muscle. The

apparent d i s c r e p a n c y

e x i s t i n g i n t h i s a r e a can p a r t l y be

e x p l a i n e d on t h e b a s i s o f c e l l u l a r h e t e r o g e n i e t y should

o f some t i s s u e s . I t

be noted t h a t smooth muscle p r e p a r a t i o n s , i n a d d i t i o n t o nerve

c e l l s and blood interstitial

v e s s e l s , contain other c e l l

types such as f i b r o b l a s t s ,

c e l l s , mast c e l l s and e n d o t h e l i a l c e l l s ( G a b e l l a G. 1982).

Because o f t h i s c e l l u l a r h e t e r o g e n i e t y

o f t h e smooth muscle

drug t r e a t m e n t may l e a d t o e l e v a t i o n o f cAMP l e v e l s i n o t h e r t y p e s i n a d d i t i o n t o smooth muscle c e l l s .

preparations cell

T h i s s i t u a t i o n might r e s u l t

-7i n erroneous

c o n c l u s i o n s because o f t h e apparent

estimated t o t a l

d i s s o c i a t i o n between'

t i s s u e cAMP l e v e l s and c o n t r a c t i l e responses

observed

i n such s t u d i e s . Some a u t h o r s t r i e d t o e x p l a i n t h e i r c o n t r o v e r s i a l

r e s u l t s by

s u g g e s t i n g c o m p a r t m e n t a l i z a t i o n o f cAMP i n t h e smooth muscle. ( F o r example, see V e s i n and Harbon, 1974).

Elucidation of a

~;

r o l e o f c y c l i c AMP-dependent p r o t e i n k i n a s e i n s e v e r a l c e l l u l a r p r o c e s s e s ; l e d t o t h e s u g g e s t i o n t h a t n o t o n l y cAMP but a l s o c y c l i c AMP-dependent p r o t e i n k i n a s e might be c o m p a r t m e n t a l i z e d

by s e l e c t i v e

a c t i v a t i o n o f hormones i n some t i s s u e s ( C o r b i n et_ a l _ , 1977;. Brunton e t a l _ , 1980).

For example, i n t h e i r c l a s s i c a l

i n t h e h e a r t , Hayes, Brunton

and Mayer (1980) observed

experiments that

i s o p r o t e r e n o l and p r o s t a g l a n d i n E-| (PGE-j) i n c r e a s e d cAMP l e v e l s and activated

the soluble protein kinase.

However, i s o p r o t e r e n o l b u t

not PGE-] produced t h e a n t i c i p a t e d a c t i v a t i o n o f p h o s p h o r y l a s e by an i n o t r o p i c a c t i o n on t h e h e a r t .

followed

A l s o i s o p r o t e r e n o l , but n o t PGE-|

a c t i v a t e d p a r t i c u l a t e p r o t e i n k i n a s e s u g g e s t i n g s p e c i f i c p o o l s o f cAMP and

i t s p r o t e i n k i n a s e might be i m p o r t a n t i n t h e e l u c i d a t i o n o f hormonal

action.

Based on these r e s u l t s and a l s o on e x t e n s i v e

e x i s t i n g i n t h e l i t e r a t u r e , Hayes and Brunton

evidence

hypothesized the

compartmentalization o f c y c l i c nucleotide a c t i o n .

In t h e i r r e c e n t

e x c e l l e n t r e v i e w a r t i c l e , (Hayes and B r u n t o n , 1982) i t was s t a t e d t h a t "Hormonally s p e c i f i c compartmentation o f cAMP o c c u r s i n c a r d i a c myocytes and p r o b a b l y i n many o t h e r c e l l

types.

The a c c u m u l a t i o n o f

cAMP a t p r e c i s e and l i m i t e d i n t r a c e l l u l a r l o c i l e a d s t o l o c a l i z e d a c t i v a t i o n o f cAMP-PK and t h e p h o s p h o r y l a t i o n o f some, b u t n o t

-8-

n e c e s s a r i l y a l l , s u b s t r a t e s o f cAMP-PK. p a r t m e n t a t i o n , we observe

As a r e s u l t o f t h i s com-

e x p e r i m e n t a l l y t h a t not a l l c e l l u l a r cAMP

i n t e r a c t s w i t h a l l endogenous cAMP-PK, and t h a t not a l l c a t a l y t i c s u b u n i t s o f cAMP-PK i n t e r a c t w i t h a l l p r o t e i n s u b s t r a t e s .

We o f f e r

t h i s as a c o r o l l a r y t o t h e p r o t e i n k i n a s e h y p o t h e s i s o f cAMP a c t i o n . " Even though v e r i f i c a t i o n o f t h i s t h e o r y i s d i f f i c u l t a t t h i s at

stage,

l e a s t some o f t h e d i s c r e p a n c i e s e x i s t i n g i n t h e smooth muscle area

w i t h r e s p e c t t o c y c l i c n u c l e o t i d e s can be e x p l a i n e d based on t h e compartmentalization

theory.

We s h o u l d not e x c l u d e t h e p o s s i b i l i t y t h a t t o some e x t e n t , t h e technical

l i m i t a t i o n s i n measurement o f t o t a l

contribute to the controversy. observed

cAMP l e v e l s might

For example, S a l a e t al_, (1979)

a d i s s o c i a t i o n between cAMP l e v e l s and s t e r o i d

i n response

t o ACTH'on s t e r o i d producing t i s s u e s .

production

However, t h i s

d i s c r e p a n c y was r e s o l v e d when they demonstrated a r i s e i n p r o t e i n k i n a s e a c t i v i t y even i n t h e apparent l e v e l s i n these t i s s u e s .

absence o f a change i n cAMP

They f u r t h e r suggested

t h a t i n s t e a d o f cAMP,

measurement o f c y c l i c AMP-dependent p r o t e i n k i n a s e (cA Kinase) might be t h e most meaningful

parameter i n d e m o n s t r a t i n g

nucleotides i n physiological

responses.

I t i s u n f o r t u n a t e t o note t h a t v e r y l i t t l e a b i l i t y o f pharmacological

a role of cyclic

i s known about t h e

agents t o c o n t r o l t h e a c t i v i t y o f cA

k i n a s e i n i n t a c t smooth muscle i n general and v a s c u l a r smooth muscle in

particular.

-9-

C.

C y c l i c AMP-Dependent P r o t e i n Kinase (cA K i n a s e ) The i n i t i a l

study o f the cA k i n a s e (Walsh e t al_,

1968)

demonstrated t h a t t h i s enzyme might be a p r i m a r y s i t e o f a c t i o n o f c y c l i c nucleotide.

Kuo arid Greengard

(1969) extended t h i s concept

by i d e n t i f y i n g t h i s enzyme i n a wide range o f mammalian t i s s u e s . T h i s u b i q u i t o u s o c c u r r e n c e o f cA k i n a s e i n d i f f e r e n t t i s s u e s i n which cAMP i s the presumed second messenger l e d t o the p r o p o s a l t h a t a m a j o r i t y o f the e f f e c t s produced by cAMP a r e mediated by a c t i v a t i o n o f protein kinase.

I t appears t h a t a c t i v a t i o n o f cA k i n a s e may

be an

o b l i g a t o r y s t e p i n t h e e x p r e s s i o n o f many hormone and d r u g - i n d u c e d responses.

T h i s enzyme has been w e l l c h a r a c t e r i z e d and the r e g u l a t o r y

mechanisms c o n t r o l l e d by t h i s p r o t e i n k i n a s e have been e x t e n s i v e l y reviewed by s e v e r a l a u t h o r s i n r e c e n t y e a r s (Walsh and Cooper, G l a s s , 1980; F l o c k h a r t and C o r b i n , 1982).

1979;

T h i s enzyme has two

p r i n c i p a l c l a s s e s o f isozymes ( C o r b i n e t al_, 10975) named type t y p e I I on the: b a s i s o f t h e i r e l u t i o n from DEAE c e l l u l o s e .

Fand-

I t has been

c o n f i r m e d t h a t the r e l a t i v e d i s t r i b u t i o n o f t h e s e two isozymes v a r i e s from s p e c i e s t o s p e c i e s and from t i s s u e t o t i s s u e .

These two

-isozymes

appear t o share a common c a t a l y t i c s u b u n i t but have d i f f e r e n t r e g u l a t o r y s u b u n i t s which might be r e s p o n s i b l e f o r t h e d i f f e r e n c e i n t h e i r p r o p e r t i e s ( C o r b i n and K e e l y , 1977).

I t was

implied

that

s p e c i f i c a c t i v a t i o n o f e i t h e r o f t h e s e isozymes might be r e s p o n s i b l e f o r the cAMP-mediated c e l l u l a r processes i n d i f f e r e n t t i s s u e s

(Schwoch,

1978; C o r b i n e t a l _ , 1977; Medieks and Hand, 1982) i n c l u d i n g smooth muscle ( G u i n o v a r t and L a r n e r , 1980; S i l v e r e t a l _ , 1982). However, f u r t h e r s t u d i e s are necessary to e l u c i d a t e s e l e c t i v e a c t i v a t i o n o f isozymes by drugs and hormones.

-10-

Agents which s t i m u l a t e t h e a c c u m u l a t i o n

o f cAMP a c t i v a t e

cA k i n a s e by t h e f o l l o w i n g mechanism ( a l s o see F i g . 1 ) .

R

2

C

2

+ 4 cAMP^-^- R

2

cAMP + 2C 4

(Inactive)

(Active)

In t h e b a s a l s t a t e o f t h e c e l l , i . e . , i n t h e absence o f hormonal s t i m u l a t i o n , cA k i n a s e e x i s t s predominantly

i n a holo-

enzyme form ( R C ) i n which a r e g u l a t o r y s u b u n i t dimer ( R ) i s bound 2

2

2

t o two c a t a l y t i c (C) s u b u n i t s .

I n t h i s form t h e enzyme i s i n a c t i v e

s i n c e t h e r e g u l a t o r y dimer e x e r t s an i n h i b i t o r y e f f e c t on t h e a c t i v i t y o f the c a t a l y t i c subunits. cytosol

When t h e cAMP c o n c e n t r a t i o n o f t h e

i n c r e a s e s , t h e n u c l e o t i d e i n t e r a c t s w i t h cAMP b i n d i n g

on t h e r e g u l a t o r y dimer and promotes t h e d i s s o c i a t i o n o f R

2

sites

from t h e

c a t a l y t i c s u b u n i t t o produce t h e a c t i v e form o f t h e enzyme ( C ) .

This

i s r e f l e c t e d i n v i t r o as an i n c r e a s e i n t h e -cAMP/+cAMP a c t i v i t y r a t i o o f t h e enzyme. the p h o s p h o r y l a t i o n

The c a t a l y t i c s u b u n i t o f cA k i n a s e c a t a l y z e s o f i n t r a c e l l u l a r enzymes and o t h e r p r o t e i n s

u s i n g ATP as t h e phosphoryl

donor.

This phosphorylation

a l t e r s the

f u n c t i o n a l a c t i v i t y o f many o f t h e enzymes i . e . , causes e i t h e r a c t i vation or inactivation.

These a c t i v i t y changes, i n t u r n , might be

r e s p o n s i b l e f o r many o f t h e p h y s i o l o g i c a l a c t i o n s o f t h e drugs. In smooth m u s c l e , microsomal p r o t e i n s ( K r a l l

e t a]_, 1978) and

myosin l i g h t c h a i n k i n a s e ( A d e l s t e i n e t al_, 1981) have been suggested t o be s u b s t r a t e s f o r c A - k i n a s e .

Phosphorylation o f these

proteins

has been i m p l i c a t e d t o be a s s o c i a t e d w i t h smooth muscle r e l a x a t i o n . Although f r e e Ca

the mechanism by which cAMP can r e g u l a t e t h e i n t r a c e l l u l a r l e v e l s i s n o t known, t h e e s s e n t i a l r o l e o f Ca

d i r e c t modulation o f actin-myosin

arid i t s

interactions i n the regulation o f

smooth muscle c o n t r a c t i o n a l s o must be c o n s i d e r e d , ( M a g a r i b u c h i Kuriyama, 1972; 1981;

D.

Webb and B h a l l a , 1976;

M a r s h a l l , 1977;

and

Webb and Bohr,

S t u l l , 1980).

F o r s k o l i n and i t s Role i n C y c l i c N u c l e o t i d e

Research

In r e c e n t y e a r s , a novel drug c a l l e d f o r s k o l i n has produced a major impact on c y c l i c n u c l e o t i d e r e s e a r c h .

Forskolin i sa cardio-

a c t i v e d i t e r p e n e d e r i v a t i v e i s o l a t e d from t h e I n d i a n p l a n t c o l e u s forskohlii.

I t has been shown i n membranes and i n t a c t c e l l s

that

f o r s k o l i n i s a potent s t i m u l a n t o f a d e n y l a t e c y c l a s e and t h e a c t i v a t i o n appears t o be r a p i d and r e v e r s i b l e (Seamon e t a l _ , 1981). e v e r , the mechanism by which f o r s k o l i n a c t i v a t e s a d e n y l a t e i s not y e t c l e a r .

Several biochemical

cyclase

studies revealed that f o r s k o l i n

has no d i r e c t e f f e c t on sodium-potassium ATPase, c y c l i c phosphodiesterase

How-

( L i n d n e r e t al_, 1 978), g u a n y l a t e

nucleotide

cyclase, cyclic

AMP-dependent p r o t e i n k i n a s e and calcium-magnesium ATPase o f s a r c o p l a s m i c r e t i c u l u m (Metzer

and L i n d n e r , 1981).

The e f f e c t s produced

by f o r s k o l i n were a l s o n o t b l o c k e d by a v a r i e t y o f r e c e p t o r

blockers

r u l i n g out a r o l e o f receptor-mediation

Seamon

and

Daly

i n i t s activation.

(1981a) r e p o r t e d t h a t f o r s k o l i n does not r e q u i r e a f u n c t i o n a l

guanine n u c l e o t i d e r e g u l a t o r y s u b u n i t t o a c t i v a t e t h e c a t a l y t i c

subunit

o f t h e enzyme and i t was suggested t h a t f o r s k o l i n might p o s s i b l y be

-12acting d i r e c t l y on the c a t a l y t i c subunit. Forskolin has been shown to increase cAMP l e v e l s in a v a r i e t y of t i s s u e s (Seamon el^aU

1981).

Several forskolin-induced c e l l u l a r

responses have been correlated to increased c y c l i c AMP s y n t h e s i s , and i t has been suggested that f o r s k o l i n may be a valuable tool in e l u c i d a t i n g the r o l e of cAMP in the physiological responses to various hormones (see for review, Seamon and Daly, 1981b). Forskolin has also been reported to relax a v a r i e t y of smooth muscles i n c l u d i n g vascular smooth muscle (Dubey et al_, 1981; Muller and Baer, 1983; Burka, 1983; Vegesntfand Diamond, 1983).

As mentioned

e a r l i e r , r e l a x a t i o n of vascular smooth muscle by B-adrenergic amines i s generally assumed to be mediated by an increase in i n t r a c e l l u l a r accumulation of c y c l i c AMP (Kukovetz ejt aj_, 1981).

If elevation of

cAMP i s responsible f o r r e l a x a t i o n of vascular smooth muscle, forskolin-induced elevation of cAMP should be correlated with r e laxation.

Evidence consistent with t h i s hypothesis has been presented

by Muller and Baer (1983).

Forskolin was capable of r e l a x i n g i s o l a t e d

preparations of rat a o r t a , bovine coronary a r t e r y , canine coronary a r t e r y , guinea"pig taenia c o l i and r a b b i t small i n t e s t i n e .

The

relaxant e f f e c t s of f o r s k o l i n on guinea pig taenia c o l i and r a b b i t small i n t e s t i n e were potentiated by phosphodiesterase inhibitors-arid f o r s k o l i n was found to d i r e c t l y stimulate adenylate cyclase from these i n t e s t i n a l smooth muscles.

Although cAMP l e v e l s were not a c t u a l l y

measured, i t was concluded from these r e s u l t s that r e l a x a t i o n of smooth muscle by f o r s k o l i n was mediated by cAMP.

However, to t h i s date, no

data c o r r e l a t i n g forskolin-induced smooth muscle r e l a x a t i o n with

-13-

i n c r e a s e s i n cAMP l e v e l s have been p u b l i s h e d . Another s t r i k i n g a s p e c t o f f o r s k o l i n e f f e c t s observed was i t s p o t e n t i a t i o n o f h o r m o n a l l y - i n d u c e d e f f e c t s on c y c l i c AMP g e n e r a t i o n . I t has been suggested t h a t low c o n c e n t r a t i o n s o f f o r s k o l i n which

alone

produce v e r y s m a l l i n c r e a s e s i n cAMP l e v e l s can g r e a t l y p o t e n t i a t e responses t o c e r t a i n hormones such as n o r e p i n e p h r i n e , i s o p r o t e r e n o l , h i s t a m i n e , PGE and VIP i n a number o f h o r m o n a l l y r e s p o n s i v e c e l l s 2

i n c l u d i n g r a t a d i p o c y t e s , r a t and guinea p i g b r a i n s l i c e s , r a t glioma c e l l s , r a t a d r e n a l c e l l s , guinea p i g t h y r o i d g l a n d s and human p l a t e l e t s (see f o r r e v i e w , Seamon and D a l y , 1981b).

The i n t r i c a t e

mechanism by which f o r s k o l i n e l i c i t s t h e tremendous p o t e n t i a t i o n o f hormonal responses

i s not y e t c l e a r .

It isstill

unknown whether o r

not f o r s k o l i n can p o t e n t i a t e t h e hormonal response i n smooth muscle. From t h e aforementioned

i n f o r m a t i o n , i t i s c l e a r t h a t , although

e l e v a t i o n o f cAMP does n o t appear t o be the p r i m a r y mechanism o f a c t i o n o f most smooth muscle r e l a x a n t s , t h e r e i s a g r e a t deal o f e v i d e n c e c o n s i s t e n t w i t h such a mechanism f o r r e l a x a t i o n o f v a s c u l a r smooth muscle by 3 - a d r e n e r g i c a g o n i s t s . dependent and

However, v e r y few s y s t e m a t i c t i m e -

dose-dependent s t u d i e s were done r e l a t i n g changes i n

cAMP l e v e l s t o r e l a x a t i o n o f v a s c u l a r smooth muscle. Hardman, 1980, f o r r e v i e w ) .

(Kramer and

E s t i m a t i o n o f c y c l i c - A M P dependent p r o t e i n

k i n a s e might f u r t h e r promote o u r u n d e r s t a n d i n g o f a r o l e o f cAMP i n t h e r e l a x a t i o n o f v a s c u l a r smooth muscle.

F o r s k o l i n may be a p o s s i b l e t o o l

to e x p l o r e t h e cAMP-mediated e f f e c t s i n smooth muscle.

I t would a l s o

be i n t e r e s t i n g t o see whether f o r s k o l i n can p o t e n t i a t e h o r m o n a l l y induced responses i n smooth muscle.

The present , study has been undertaken t o answer some o f 1

these questions.

Attempts have been made t o c o r r e l a t e changes i n

cAMP l e v e l s and p r o t e i n - k i n a s e

a c t i v i t y w i t h r e l a x a t i o n o f bovine

c o r o n a r y a r t e r i e s by f o r s k o l i n .

The e f f e c t s o f f o r s k o l i n were

compared t o those o f i s o p r o t e r e n o l , a B - a d r e n e r g i c a g o n i s t generally

believed

to increase

t o r e l a x smooth muscles by v i r t u e o f i t s a b i l i t y

c y c l i c AMP l e v e l s .

-15-

SPECIFIC

1.

To

GOALS

OF

THE

PRESENT

study time-dependent and

on c y c l i c AMP a r t e r i e s and

l e v e l s and

INVESTIGATION

dose-dependent e f f e c t s o f f o r s k o l i n

tension

i n i s o l a t e d bovine c o r o n a r y

to determine whether a c o r r e l a t i o n e x i s t s between

e l e v a t i o n o f cAMP and

r e l a x a t i o n o f the v a s c u l a r

smooth muscle..

These e f f e c t s would be compared w i t h those o f i s o p r o t e r e n o l , 3-adrenergic 2.

To

agonist.

see whether low doses o f f o r s k o l i n can

t e r e n o l -induced e f f e c t s i n v a s c u l a r

potentiate

isopro-

smooth m u s c l e , as i s the

case i n o t h e r t i s s u e s . 3.

To e s t a b l i s h o p t i m a l c o n d i t i o n s

f o r the e s t i m a t i o n

enzyme c y c l i c AMP-dependent p r o t e i n

of

the

kinase i n bovine coronary

arteries. 4.

To

s t u d y the e f f e c t s o f f o r s k o l i n on c y c l i c AMP-dependent

protein

k i n a s e a c t i v i t y and

tension

compare these e f f e c t s w i t h those o f

i n coronary a r t e r i e s isoproterenol.

and

a

-16-

MATER.IALS AND METHODS

A.

MATERIALS The f o l l o w i n g c h e m i c a l s were purchased from Sigma Chemical

Co.: sodium c h l o r i d e , p o t a s s i u m c h l o r i d e , magnesium c h l o r i d e , magnesium s u l p h a t e , c a l c i u m c h l o r i d e , sodium b i c a r b o n a t e , sodium phosphate monobasic, g l u c o s e , t r i c h l o r o a c e t i c - a c i d , T r i t o n X-100, Trizma. adenosine t r i p h o s p h a t e ( T r i s ATP), p o t a s s i u m phosphate, ethylenediamine

t e t r a a c e t i c a c i d (EDTA), methyl

isobutylxanthine,

d i t h i o t h r e i t o l , H i s t o n e 11-A, sodium f l u o r i d e , c y c l i c AMP, serum albumin and Whatman 3MM

bovine

(-)-isoproterenol. (GF/A) f i l t e r papers (2.3 cm d i a m e t e r ) and 5 ml

s c i n t i l l a t i o n v i a l s were o b t a i n e d from Western S c i e n t i f i c ACS s c i n t i l l a t i o n f l u i d and f r - p ] ATP 3 2

Co.

(20 ci/mMole) were

purchased from Amer sham.F o r s k o l i n was o b t a i n e d from Calbiochem. o f f o r s k o l i n was prepared i n 95% e t h a n o l .

A ImM

stock s o l u t i o n

T h i s s o l u t i o n was

w i t h w a t e r and added t o the muscle baths t o g i v e the d e s i r e d concentration.

diluted final

C o n t r o l s t r i p s were t r e a t e d w i t h t h e a p p r o p r i a t e

concentration of ethanol. C y c l i c - A M P radio-immunoassay

k i t s were purchased from Becton

D i c k i n s o n Canada L t d . Fresh bovine h e a r t s were o b t a i n e d from a l o c a l

slaughter

house ( I n t e r c o n t i n e n t a l P a c k e r s ) f o r t h i s e n t i r e s t u d y .

-17-

B. I.

METHODS

P r e p a r a t i o n o f muscle samples Bovine h e a r t s were o b t a i n e d

immediately

a f t e r s l a u g h t e r , immersed

i n i c e c o l d r e g u l a r Krebs s o l u t i o n and t r a n s p o r t e d t o t h e l a b o r a t o r y . Branches o f t h e l e f t a n t e r i o r d e s c e n d i n g and c i r c u m f l e x a r t e r i e s were d i s s e c t e d f r e e and h e l i c a l

coronary

s t r i p s were p r e p a r e d .

s t r i p s were suspended a t 37°C under 2 g t e n s i o n i n Krebs s o l u t i o n with the f o l l o w i n g composition MgS0 , 2.33; C a C l , 1.26; NaHC0 4

2

3>

The

bicarbonate

(mM): N a C l , 118; K C l , 5.7;

2.5; N a H P 0 , 1.17 and g l u c o s e , 11. 2

4

S o l u t i o n s were a e r a t e d w i t h 5% C 0 and 95% 0 , which m a i n t a i n e d 2

at approximately

2

7.4. A l t e r a t i o n s i n tone were m o n i t o r e d

using force transducers.

t h e pH

isometrically

A f t e r a 2 hour e q u i l i b r i u m p e r i o d , t h e s t r i p s

were i n i t i a l l y c o n t r a c t e d by r e p l a c i n g t h e b a t h i n g s o l u t i o n w i t h one c o n t a i n i n g 124 fiiM K C l . A f t e r repeated

washings w i t h normal b u f f e r ,

the s t r i p s were r e c o n t r a c t e d s u b m a x i m a l l y by N a p o l i e t al_.

(1980)(see

w i t h 30 mM KCl as d e s c r i b e d

F i g s . 9 and 1 0 ) .

When t h e s t r i p s had

a t t a i n e d a steady s t a t e l e v e l o f t e n s i o n i n 30 mM K C l , t e s t drugs were added d i r e c t l y t o t h e muscle b a t h s , and t h e muscle s t r i p s were q u i c k f r o z e n a t p r e d e t e r m i n e d times by clamping them w i t h a p a i r o f tongs precooled

i n l i q u i d nitrogen.

-80°C u n t i l assays.

The f r o z e n samples were s t o r e d a t

used f o r c y c l i c AMP and c y c l i c AMP-dependent p r o t e i n k i n a s e

T e n s i o n and c y c l i c AMP o r c y c l i c AMP-dependent p r o t e i n k i n a s e

were t h u s determined i n t h e same muscle s t r i p s .

11.

Measurement o f c y c l i c AMP C y c l i c AMP l e v e l s i n t h e f r o z e n t i s s u e s were determined u s i n g

radio-irrniunoas'say k i t s s u p p l i e d by Beckton D i c k i n s o n

Canada, I n c .

B r i e f l y , t h e f r o z e n samples were homogenized i n 6% t r i c h l o r o a c e t i c a c i d , the t r i c h l o r o a c e t i c a c i d removed by e t h e r e x t r a c t i o n and r a d i o immunoassay performed on t h e aqueous e x t r a c t s .

Results are expressed

as pmoles o f c y c l i c AMP per g wet w e i g h t o f t i s s u e .

Preparation

o f e x t r a c t s and assay o f c y c l i c AMP-dependent p r o t e i n

kinase

A p p r o x i m a t e l y 100-200 mg o f f r o z e n t i s s u e was suspended a t 4®C i n 10 volumes o f b u f f e r (pH 6.8) c o n t a i n i n g lOmM potassium phosphate, 10 mM EDTA, 0.5 mM methyl i s o b u t y l x a n t h i n e and 0.5 mM 1 , 4 - d i t h i o t h r e i t o l , w i t h and w i t h o u t 0.5 M NaCl as suggested by S i l v e r e t a l _ .

(1982).

The

t i s s u e was homogenized w i t h a P o l y t r o n homogenizer ( s e t t i n g t h e speed a t 8 f o r 30 seconds) and Mhe homogenate was i m m e d i a t e l y c e n t r i f u g e d a t 30,000 x g f o r 15 min ( S o r v a l l , RC-2B c e n t r i f u g e ) a t 4°C t o form s o l u b l e ( s u p e r n a t a n t ) and p a r t i c u l a t e ( p e l l e t ) f r a c t i o n s . n a t a n t f r a c t i o n was i m m e d i a t e l y assayed f o r p r o t e i n k i n a s e

The s u p e r activity.

When a s s a y e d , t h e p e l l e t c o n t a i n i n g t h e p a r t i c u l a t e f r a c t i o n was g e n t l y washed t w i c e w i t h 10 volumes o f homogenizing b u f f e r t o remove l o o s e l y adhering m a t e r i a l .

The washed p e l l e t was homogenized w i t h a

hand homogenizer i n 4 volumes o f homogenizing b u f f e r c o n t a i n i n g 0.1% T r i t o n X-100.

A f t e r homogem'zation, t h e sample was kept on i c e f o r

10 min and c e n t r i f u g e d a t 30,000 x g f o r 15 min. The s u p e r n a t a n t f r a c t i o n from t h e T r i t o n - t r e a t e d p e l l e t was used t o e s t i m a t e p a r t i c u l a t e protein kinase The

activity.

p r o t e i n k i n a s e a c t i v i t y was determined by measuring t h e

32 32 t r a n s f e r o f :-.p from £Y~ p] ATP t o h i s t o n e i n t h e presence and absence v

-19-

of

2 yM cAMP as d e s c r i b e d by C o r b i n and Reiman (1975).

The a s s a y

r e a c t i o n was s t a r t e d by a d d i n g 20 y l o f t h e s u p e r n a t a n t t o 50 y l o f the r e a c t i o n m i x t u r e c o n t a i n i n g 20 mM potassium phosphate (pH 6 . 8 ) , 100 yM

[ t r p ] ATP (50-100 cpm/pmol), 10 mM magnesium c h l o r i d e , 3 2

100 yg h i s t o n e I I - A (Sigma), and 10 mM sodium f l u o r i d e , i n t h e presence and absence o f 2 yM c y c l i c AMP. for

The i n c u b a t i o n was c a r r i e d o u t a t 30°C

10 min. The r e a c t i o n was t e r m i n a t e d by p i p e t t i n g 60' y l a l i q u o t s o f

the r e a c t i o n m i x t u r e on t d f i l t e r paper d i s c s (Whatmann 3 mm, 2.3 cm d i a m e t e r ) which were i m m e d i a t e l y dropped acetic acid.

i n t o 10% i c e c o l d

trichloro-

The f i l t e r papers were washed 4 times (15 min each) i n

t r i c h l o r o a c e t i c a c i d f o l l o w e d by one 5 min wash i n 95% e t h a n o l and one in ether.

The f i l t e r papers were then d r i e d and t r a n s f e r r e d i n t o .:

scintillation vials.

These v i a l s were f i l l e d w i t h ACS s c i n t i l l a t i o n

f l u i d (5 m l ) and t h e r a d i o a c t i v i t y counted i n a MARK I I I s c i n t i l l a t i o n counter.

The p r o t e i n k i n a s e a c t i v i t y was e x p r e s s e d as pmoles phosphate

t r a n s f e r r e d per mg p r o t e i n .

The e x t e n t o f c y c l i c AMP-dependent p r o t e i n

k i n a s e a c t i v a t i o n i n t h e s u p e r n a t a n t was a s s e s s e d by c a l c u l a t i n g t h e a c t i v i t y r a t i o , which i s t h e r a t i o o f k i n a s e a c t i v i t y i n t h e absence o f added c y c l i c AMP t o t h a t i n t h e presence o f enough c y c l i c AMP t o f u l l y a c t i v a t e t h e enzyme.

IV.

Protein determination The c o n c e n t r a t i o n o f p r o t e i n i n t h e s u p e r n a t a n t and t h e p e l l e t was determined

a c c o r d i n g t o t h e Lowry procedure

bovine serum albumin as a s t a n d a r d .

(Lowry e j t a l _ . , 1951) u s i n g

-20-

V.

Statistical

Analyses:

Data were e v a l u a t e d s t a t i s t i c a l l y by means o f a s t u d e n t ' s t t e s t . A p r o b a b i l i t y o f l e s s than 0.05 significance.

was a c c e p t e d as the l e v e l o f

statistical

RESULTS

CONDITIONS FOR THE ASSAY OF CYCLIC AMP-DEPENDENT PROTEIN KINASE (CA KINASE) IN INTACT TISSUE

An attempt was made t o s t a n d a r d i z e t h e p r o t e i n k i n a s e assay c o n d i t i o n s i n o u r l a b o r a t o r y .

The f o l l o w i n g experiments

were undertaken t o determine optimum c o n d i t i o n s f o r t h e assay.

1)

L i n e a r i t y with time:

The e f f e c t o f i n c u b a t i o n time on

the p r o t e i n k i n a s e a c t i v i t y i n c o r o n a r y A typical

a r t e r i e s was examined.

experiment i s shown i n F i g . 2.

The experiment method

was t h e same as d e s c r i b e d e a r l i e r except t h e i n c u b a t i o n time was varied.

The r e a c t i o n r a t e was l i n e a r a t 30°C up t o 40 min i n

the presence o f 2 uM cAMP and up t o 20 min i n t h e absence o f cAMP-

The p r o t e i n k i n a s e a c t i v i t y r a t i o remained c o n s t a n t up

t o 20 min ( d a t a n o t shown). 2)

L i n e a r i t y with protein concentrations:

The e f f e c t o f enzyme

c o n c e n t r a t i o n on t h e p r o t e i n k i n a s e a c t i v i t y i s shown i n F i g . 3. D i l u t i o n o f the supernatant genization buffer.

f r a c t i o n was made w i t h t h e homo-

The p r o t e i n k i n a s e a c t i v i t y i n t h i s

was determined as d e s c r i b e d under e x p e r i m e n t a l Histone phosphorylation

by t h e c o r o n a r y

fraction

procedures.

a r t e r y p r o t e i n k i n a s e was

-22-

l i n e a r with respect

to the amount o f enzyme added up to 30

o f p r o t e i n per assay. up to t h i s 3)

P r o t e i n kinase

r a t i o remained c o n s t a n t

concentration.

E f f e c t o f magnesium c h l o r i d e on cA k i n a s e a c t i v i t y :

assay was

run

under r o u t i n e c o n d i t i o n s

the e x c e p t i o n

that M g

+ +

concentration

t h a t maximum p r o t e i n k i n a s e MgCl2 and

yg

The

s t a t e d i n methods w i t h

was

varied.

a c t i v i t y was

F i g . 4 shows

observed a t 15-20

f u r t h e r i n c r e a s i n g the c o n c e n t r a t i o n

mM

i n h i b i t e d the

enzyme a c t i v i t y . 4)

E f f e c t o f NaF

preparations

may

on p r o t e i n k i n a s e a c t i v i t y : c o n t a i n ATPases and

the e f f e c t s o f these ATPases. presence o f 2 yM cAMP. described

e a r l i e r e x c e p t NaF

concentration

was

k i n a s e a c t i v i t y was

o f NaF

i n h i b i t e d the k i n a s e

activity.

E f f e c t o f T r i t o n X-100

minimizes

Assays were performed i n the

lower c o n c e n t r a t i o n s

kinase a c t i v i t y :

a d d i t i o n o f NaF

E x p e r i m e n t a l procedure was

shown i n F i g . 5, the t o t a l

5)

Crude enzyme

(10-20 mM)

concentration

Frozen a r t e r i a l

same as

varied.

As

not changed by

but h i g h e r

concentrations

on p a r t i c u l a t e p r o t e i n

s t r i p s were homogenized i n the

normal b u f f e r , the homogenate d i v i d e d i n t o s e v e r a l a l i q u o t s

and

p a r t i c u l a t e f r a c t i o n s ( p e l l e t s ) o f these a l i q u o t s were prepared as d e s c r i b e d

i n the methods.

Then the p e l l e t s were suspended

i n the b u f f e r c o n t a i n i n g d i f f e r e n t c o n c e n t r a t i o n s and

centrifuged.

The

of T r i t o n

s u p e r n a t a n t f r a c t i o n s from t h e s e T r i t o n -

t r e a t e d p e l l e t s were used t o e s t i m a t e p a r t i c u l a t e p r o t e i n activity.

The

X-100

kinase

assays were run under r o u t i n e c o n d i t i o n s , i n the

-23-

presence and absence o f 2 uM cAMP i n t h e f i n a l mixture. low

incubation

As shown i n F i g . 6, t h e a c t i v i t y o f t h e p e l l e t was

i n t h e absence o f T r i t o n X-100, and i n c r e a s e d

t o a maximum

a t 0.2%, i n d i c a t i n g s o l u b i l i z a t i o n o f t h e enzyme by T r i t o n . higher the

concentrations,

At

p r o t e i n k i n a s e a c t i v i t y was i n h i b t e d by

detergent. In summary, t h e f o l l o w i n g s t a n d a r d c o n d i t i o n s were used:

The assay was r o u t i n e l y run f o r 10 min a t 30°C, w i t h 10 mM M g C l , 2

10 mM NaF and 20 u l o f t h e s u p e r n a t a n t c o n t a i n i n g in the f i n a l

incubation mixture.

T r i t o n X-100 was i n c l u d e d

10-25 ug

protein

In t h e p e l l e t e x p e r i m e n t s , 0.1%

i n t h e homogenization b u f f e r used f o r

suspending t h e p e l l e t .

B.

TIME COURSE STUDIES OF ISOPROTERENOL AND FORSKOLIN ON CYCLIC AMP LEVELS AND TENSION IN BOVINE CORONARY ARTERIES Time c o u r s e s f o r e l e v a t i o n o f c y c l i c AMP and r e l a x a t i o n o f b o v i n e c o r o n a r y a r t e r i e s by 1 uM i s o p r o t e r e n o l This concentration with respect

a r e shown i n F i g . 7.

o f i s o p r o t e r e n o l was a m a x i m a l l y - e f f e c t i v e

to relaxation.

C y c l i c AMP l e v e l s were s i g n i f i c a n t l y

i n c r e a s e d w i t h i n 1 minute a f t e r a d d i t i o n o f i s o p r o t e r e n o l

to the

muscle b a t h , which was t h e e a r l i e r time p o i n t a t which muscle r e l a x a t i o n c o u l d be d e t e c t e d . increased

C y c l i c AMP l e v e l s were f u r t h e r

a t 4 and 10 minute p o i n t s .

o c c u r r e d w i t h i n 10 minutes.

one

Maximal r e l a x a t i o n had

-24-

A s i m i l a r experiment with,a m a x i m a l l y - r e l a x a n t c o n c e n t r a t i o n o f f o r s k o l i n i s shown i n F i g . 8.

A t 1 minute a f t e r a d d i t i o n o f f o r s k o l i n

t o t h e b a t h , no r e l a x a t i o n and no c y c l i c AMP e l e v a t i o n were seen. A t 2 m i n u t e s , c y c l i c AMP l e v e l s had i n c r e a s e d s i g n i f i c a n t l y , and t h e muscles had begun t o r e l a x .

W i t h i n 8 m i n u t e s , c y c l i c AMP l e v e l s had

i n c r e a s e d a p p r o x i m a t e l y 30 f o l d , and t h e muscles had c o m p l e t e l y relaxed.

C.

EFFECTS OF VARIOUS CONCENTRATIONS OF ISOPROTERENOL AND

FORSKOLIN

ON cAMP LEVELS AND TENSION IN CORONARY ARTERIES The e f f e c t s o f v a r i o u s c o n c e n t r a t i o n s o f i s o p r o t e r e n o l and f o r s k o l i n on c y c l i c AMP l e v e l s and t e n s i o n i n b o v i n e c o r o n a r y a r e shown i n T a b l e I I . Both drugs caused i n c r e a s e s i n c y c l i c AMP l e v e l s .

arteries

concentration-dependent

However, a t lower c o n c e n t r a t i o n s o f

f o r s k o l i n (0.01-0.1 y M ) , marked i n c r e a s e s i n c y c l i c AMP l e v e l s were observed w i t h no muscle r e l a x a t i o n . A d d i t i o n o f 1 yM i s o p r o t e r e n o l i n c o m b i n a t i o n w i t h 10 yM f o r s k o l i n i n c r e a s e d c y c l i c AMP l e v e l s by a p p r o x i m a t e l y 360 f o l d ( T a b l e I I ) . T h i s i s much g r e a t e r than t h e sum o f t h e i n c r e a s e s p r o duced by i s o p r o t e r e n o l o r f o r s k o l i n a l o n e (3.7 and 30 f o l d , r e s p e c t i v e l y ) . . The c o m b i n a t i o n r e l a x e d t h e muscles by 111% ( i . e . , t h e K C l - i n d u c e d tone was c o m p l e t e l y e l i m i n a t e d , and a p a r t i a l r e s t i n g tone a l s o o c c u r r e d ) .

r e l a x a t i o n o f the 2 g

(See F i g . 1.0, f o r e x p e r i m e n t a l p r o t o c o l )

-25-

D.

EFFECTS OF ON

FORSKOLIN AND

CYCLIC AMP

The

LEVELS AND

e f f e c t s o f 0.1

e l e v a t i o n and

ISOPROTERENOL, ALONE AND

uM

TENSION IN BOVINE CORONARY ARTERIES

and

10 uM

f o r s k o l i n on the c y c l i c

the r e l a x a t i o n caused by two

i s o p r o t e r e n o l are shown i n F i g u r e ; ! ! . c y c l i c AMP manner.

l e v e l s and

The

IN COMBINATION,

concentrations

Isoproterenol

AMP

of

alone

increased

r e l a x e d the c o r o n a r y a r t e r i e s i n a dose-dependent

lower c o n c e n t r a t i o n

o f f o r s k o l i n (0.1 uM)

did

not

p o t e n t i a t e t h e e f f e c t s o f e i t h e r dose o f i s o p r o t e r e n o l

on c y c l i c

l e v e l s or t e n s i o n .

f o r s k o l i n plus

0.1

uM

In f a c t , the c o m b i n a t i o n o f 0.1

isoproterenol

increased

f o r s k o l i n a l o n e ( F i g u r e 11). (10 uM)

c y c l i c AMP

on c y c l i c AMP

l e v e l s l e s s than d i d 0.1

the e f f e c t s o f both c o n c e n t r a t i o n s l e v e l s ( F i g u r e 11).

The

f o r s k o l i n was

much g r e a t e r than the sum

agent a l o n e .

The

concentration

complete r e l a x a t i o n o f the a r t e r i e s .

of

magnitude o f

i n c r e a s e caused by a c o m b i n a t i o n o f i s o p r o t e r e n o l

10 uM

uM

However, the h i g h e r dose o f f o r s k o l i n

markedly p o t e n t i a t e d

isoproterenol

c y c l i c AMP

uM

AMP

and

the 10

uM

o f the increases' caused by e i t h e r

o f f o r s k o l i n a l o n e caused a l m o s t T h e r e f o r e , under t h e s e c o n d i t i o n s ,

we were unable to demonstrate a p o t e n t i a t i o n o f the r e l a x a n t e f f e c t s o f isoproterenol. The

F i g s . 9 and

10, f o r e x p e r i m e n t a l

protocol).

above r e s u l t s i n d i c a t e t h a t p o t e n t i a t i o n o f the e f f e c t s o f

isoproterenol 10 uM

(See

on c y c l i c AMP

l e v e l s i n these preparations

f o r s k o l i n but not w i t h 0.1

concentration proterenol

yM.

A c l o s e r approximation, o f

o f f o r s k o l i n needed t o p o t e n t i a t e the e f f e c t s o f

i s provided

with

the iso-

i n the r e s u l t s o f the experiment shown i n

F i g u r e 12.

The

1 and

f o r s k o l i n on c y c l i c AMP

10 uM

occurred

e f f e c t s o f 0.1

uM

isoproterenol l e v e l s and

t o g e t h e r w i t h 0.01, tension i n coronary

0.1,

-26-

a r t e r i e s are shown i n the f i g u r e .

The

were l e s s than a d d i t i v e a t 0.01

f o r s k o l i n , approximately a d d i t i v e

at TyM

yM

e f f e c t s on c y c l i c AMP

f o r s k o l i n . Therefore, potentiation occurred only with

c e n t r a t i o n s above 1 yM.

increased

c y c l i c AMP

con-

I t s h o u l d be noted t h a t , as p r e v i o u s l y shown

i n T a b l e I I , l o w e r doses o f f o r s k o l i n a l o n e (0.01-OH yM)

E.

levels

significantly

l e v e l s (2-5 f o l d ) but d i d not r e l a x the a r t e r i e s .

ACTIVATION OF CYCLIC AMP-DEPENDENT PROTEIN KINASE IN BOVINE CORONARY ARTERIES BY

ISOPROTERENOL AND

FORSKOLIN

S i l v e r e t a l _ . (1982) have r e c e n t l y r e p o r t e d bovine c o r o n a r y a r t e r i e s by i s o p r o t e r e n o l

i s associated with a c t i v a t i o n

o f s o l u b l e c y c l i c AMP-dependent p r o t e i n k i n a s e . i t was

n e c e s s a r y t o add

0.5

They a l s o r e p o r t e d

that

M NaCl to the homogenization medium i n o r d e r

t o demonstrate i s o p r o t e r e n o l - i n d u c e d preparations.

that r e l a x a t i o n of

a c t i v a t i o n o f the k i n a s e

i n these

S i m i l a r c o n d i t i o n s were used, i n the p r e s e n t s t u d y , t o

compare the e f f e c t s o f i s o p r o t e r e n o l

and

f o r s k o l i n on s o l u b l e

cyclic

AMP-dependent p r o t e i n k i n a s e a c t i v i t y i n bovine c o r o n a r y a r t e r i e s .

The

e f f e c t s of several concentrations

the

o f f o r s k o l i n and

k i n a s e a c t i v i t y measured w i t h and w i t h o u t 0.5 t i o n b u f f e r are p r e s e n t e d i n T a b l e I I I .

isoproterenol

M NaCl i n the

When 0.5

M NaCl was

i n the homogenization b u f f e r , no s i g n i f i c a n t i n c r e a s e

o r w i t h 0.1

yM

homogenizanot

i n kinase

r a t i o s were observed i n c o r o n a r y a r t e r i e s t r e a t e d w i t h lyM

on

included activity

isoproterenol

f o r s k o l i n . T h i s i s i n s p i t e o f the f a c t t h a t c y c l i c

l e v e l s were s i g n i f i c a n t l y i n c r e a s e d

by these c o n c e n t r a t i o n s

(see F i g u r e 1 1 ) .

M NaCl was

However, when 0.5

AMP

o f the drugs

added t o the homogeniza-

t i o n b u f f e r as suggested by S i l v e r ejt a l _ . ( 1 9 8 2 ) , k i n a s e a c t i v i t y r a t i o s

-27-

were found t o be s i g n i f i c a n t l y i n c r e a s e d ( T a b l e I I I ) .

Activity

r a t i o s i n c o n t r o l p r e p a r a t i o n s were a l s o i n c r e a s e d under these conditions.

These r e s u l t s t h e r e f o r e agree w i t h those o f S i l v e r

e t al_. (1982) f o r i s o p r o t e r e n o l . Higher doses o f f o r s k o l i n , which produced v e r y marked i n c r e a s e s i n c y c l i c AMP

levels,

significantly

i n c r e a s e d p r o t e i n k i n a s e a c t i v i t y r a t i o s even i n the absence o f 0.5 NaCl i n the homogenization medium (Table 3 ) .

A combination

f o r s k o l i n and 1 yM i s o p r o t e r e n o l r e s u l t e d i n e x t r e m e l y l e v e l s o f c y c l i c AMP

o f 10

M yM

high t i s s u e

( F i g u r e 11) and caused a l m o s t maximal

o f c y c l i c AMP-dependent p r o t e i n k i n a s e w i t h and w i t h o u t 0.5

activation M NaCl

i n the homogenization medium.

F.

EFFECT OF NaCl ON SOLUBLE PROTEIN KINASE ACTIVITY I t can be seen from T a b l e 3 t h a t t o t a l

sured i n the presence o f 2 yM c y c l i c AMP) 60% i n the high NaCl e x p e r i m e n t s . a c t i v i t i e s were a f f e c t e d . e t al_. ( 1 9 8 2 ) , who

kinase a c t i v i t i e s

(mea-

were i n h i b i t e d by as much as

C o n t r o l as w e l l as d r u g - t r e a t e d

T h i s i s i n c o n t r a s t t o the r e s u l t s o f S i l v e r

noted t h a t the a d d i t i o n o f 0.5

M NaCl to the homo-

g e n i z a t i o n medium d i d not cause any s i g n i f i c a n t changes i n t o t a l activity.

kinase

We t h e r e f o r e d e c i d e d t o f u r t h e r i n v e s t i g a t e the e f f e c t o f

NaCl on the s o l u b l e k i n a s e a c t i v i t y by measuring c o n t r o l and

hormonally-

s t i m u l a t e d c y c l i c AMP-dependent p r o t e i n k i n a s e a c t i v i t y i n the presence o f i n c r e a s i n g c o n c e n t r a t i o n s o f NaCl.

As shown i n F i g u r e 13, i n c r e a s i n g

the c o n c e n t r a t i o n o f NaCl i n the homogenization medium caused a p r o g r e s s i v e decrease i n t o t a l

kinase a c t i v i t y i n c o n t r o l preparations.

c o n s i s t e n t changes i n basal a c t i v i t y were seen.

S i m i l a r r e s u l t s were

No

-28-

a l s o obtained 14).

i n muscles t r e a t e d w i t h 1 yM

When NaCl was

i s o p r o t e r e n o l . (See

removed from the s o l u t i o n s by d i a l y s i n g f o r 2 hr

against r e g u l a r homogenization b u f f e r , enzyme a c t i v i t y was toward c o n t r o l l e v e l s ( F i g u r e 15).

t h i s p r o b a b l y accounts f o r the i n c r e a s e d our experiments under these

the

kinase a c t i v i t y

and

a c t i v i t y r a t i o s observed i n

conditions.

EFFECT OF ADDITION OF CYCLIC AMP AND

returned

Thus, a d d i t i o n o f NaCl t o

homogenization medium appeared t o i n h i b i t t o t a l

G.

Figure

TO THE

HOMOGENATE ON

SUPERNATANT

PARTICULATE PROTEIN KINASE ACTIVITY

I t can be seen from the l e f t hand panel o f T a b l e 3 t h a t drugs which caused l a r g e i n c r e a s e s decreases i n t o t a l

i n t i s s u e c y c l i c AMP

c y c l i c AMP-dependent p r o t e i n k i n a s e

e s t i m a t e d i n the absence o f added N a C l . s u p e r n a t a n t may

l e v e l s a l s o caused

be due

activity

T h i s decreased a c t i v i t y i n the

t o t r a n s l o c a t i o n o f the enzyme from the

t o the p a r t i c u l a t e f r a c t i o n .

S i m i l a r observations

been noted by K r a l l ejt a l _ . (1978) and

soluble

have p r e v i o u s l y

Harbon e t a l _ . (1978) i n r a t

myometrium t r e a t e d w i t h c a t e c h o l a m i n e s , s u g g e s t i n g t h a t t r a n s l o c a t i o n o f the enzyme t o a membrane f r a c t i o n might have o c c u r r e d i n t h a t t i s s u e as w e l l . In o r d e r t o f u r t h e r i n v e s t i g a t e t h i s p o s s i b i l i t y , f r o z e n s t r i p s were homogenized, the homogenate d i v i d e d i n t o two 2 yM c y c l i c AMP o f c y c l i c AMP

added t o one

o f them.

a l i q u o t s , and

As shown i n F i g u r e 15,

t o the homogenization b u f f e r decreased the t o t a l

a c t i v i t y i n the s u p e r n a t a n t f r a c t i o n .

This

i s s i m i l a r t o the

o b s e r v e d : i n a r t e r i e s i n which high endogenous c y c l i c AMP

arterial

addition kinase decreases

l e v e l s had

-29-

been induced by drug t r e a t m e n t (see T a b l e I I I ) . a c t i v i t y was determined

When the k i n a s e

i n the p a r t i c u l a t e f r a c t i o n o f t h e s e

as d e s c r i b e d i n Methods, i t was

muscles

found t h a t the decrease i n t o t a l

k i n a s e . a c t i v i t y i n t h e s u p e r n a t a n t was accompanied by a c o r r e s p o n d i n g i n c r e a s e i n a c t i v i t y i n the p e l l e t ( F i g u r e 16). a c t i v i t y (measured i n the absence o f c y c l i c AMP

Basal c y c l i c

AMP

i n the f i n a l i n c u b a t i o n

m i x t u r e ) i s a l s o i n c r e a s e d i n the p e l l e t o b t a i n e d from p r e p a r a t i o n s homogenized i n t h e presence o f 2 yM c y c l i c AMP.

This altered

distribu-

t i o n o r t r a n s l o c a t i o n o f k i n a s e a c t i v i t y was a l s o observed even i f the c o r o n a r y a r t e r i e s were homogenized i n b u f f e r c o n t a i n i n g 0.5 M NaCl ( d a t a not shown).

H.

EFFECTS OF ISOPROTERENOL AND IN SUPERNATANT AND RELAXATION OF THE

FORSKOLIN ON PROTEIN KINASE ACTIVITY

PARTICULATE FRACTIONS.

RELATIONSHIP"TO

CORONARY ARTERIES

As shown e a r l i e r (see F i g u r e 7 ) , r e l a x a t i o n o f c o r o n a r y a r t e r i e s by i s o p r o t e r e n o l was c o r r e l a t e d i n a time-dependent manner w i t h e l e v a t i o n o f c y c l i c AMP c y c l i c AMP

i n the t i s s u e s , . S i m i l a r l y , i n the p r e s e n t

l e v e l s were s i g n i f i c a n t l y e l e v a t e d a f t e r a 1 min exposure

1 yM i s o p r o t e r e n o l , a t which time the muscles (Table IV).

experiment, to

had j u s t begun t o r e l a x

A f t e r a 10 min exposure t o the i s o p r o t e r e n o l , c y c l i c

AMP

l e v e l s were f u r t h e r e l e v a t e d and t h e muscles were m a x i m a l l y r e l a x e d . Even though c y c l i c AMP

l e v e l s were e l e v a t e d i n t h e s e p r e p a r a t i o n s , no

a c t i v a t i o n o f the p r o t e i n k i n a s e i n t h e s u p e r n a t a n t f r a c t i o n s observed i f NaCl was o m i t t e d from the homogenization Table I I I ) .

was

( T a b l e IV, see a l s o

However, when the p r o t e i n k i n a s e a c t i v i t y was

estimated i n

-30-

the p a r t i c u l a t e f r a c t i o n s from these same a r t e r i e s , a s i g n i f i c a n t i n c r e a s e i n a c t i v i t y was As was AMP

observed even a t the 1 min

noted e a r l i e r , 0.1

yM

time ( T a b l e

IV).

f o r s k o l i n s i g n i f i c a n t l y increased c y c l i c

l e v e l s w i t h o u t p r o d u c i n g any

r e l a x a t i o n o f the a r t e r i e s .

In

the

p r e s e n t e x p e r i m e n t s , t h i s dose o f f o r s k o l i n a l s o s i g n i f i c a n t l y i n c r e a s e d the relaxation

particulate protein

(Table IV).

H i g h e r doses o f f o r s k o l i n (1-10

l a r g e r i n c r e a s e s i n c y c l i c AMP stimulated

soluble

and

kinase a c t i v i t y without causing

l e v e l s , relaxed

particulate protein

yM)

produced

the m u s c l e s , and

kinase a c t i v i t y .

also

-31-

F i g u r e 2: The e f f e c t o f i n c u b a t i o n time on c y c l i c AMPdependent p r o t e i n k i n a s e a c t i v i t y i n c o r o n a r y arteries.

P r o t e i n k i n a s e a c t i v i t y was measured

a t d i f f e r e n t time p o i n t s , both i n t h e presence and absence o f 2 yM cAMP in.:;the f i n a l mixture.

incubation

Values r e p r e s e n t mean o f 2 e x p e r i m e n t s .

-32-

f o x r a r c AMP (N.2) 5500

4400

UJ

LS

I

3300

UJ

QC

LU U_

z S tn

2200

LU —J o a.

UOO

20 30 TIME (MINS)

-33-

F i g u r e 3: The e f f e c t o f enzyme c o n c e n t r a t i o n on c y c l i c - A M P dependent p r o t e i n k i n a s e a c t i v i t y i n c o r o n a r y Supernatant

f r a c t i o n was d i l u t e d w i t h

arteries.

homogenization

b u f f e r and p r o t e i n k i n a s e a c t i v i t y e s t i m a t e d i n t h e presence

and absence o f 2 yM cAMP i n t h e f i n a l i n -

cubation mixture. experiments.

Values r e p r e s e n t mean o f 2 s i m i l a r

-34-

(°) (-)CflMP (N*2) (*) C+JCflMP (N«2)

PROTEIN CONC (MG)

-35-

F i g u r e 4: The e f f e c t s o f magnesium c h l o r i d e on c y c l i c AMP-dependent p r o t e i n k i n a s e a c t i v i t y i n coronary a r t e r i e s .

Protein kinase a c t i v i t y

e s t i m a t e d i n t h e presence cAMP i n t h e f i n a l

and absence o f 2 yM

incubation mixture.

r e p r e s e n t mean o f 2 s i m i l a r

Values

experiments.

-36-

(x) (-) CAMP (N-2) (a) (+) CAMP (N-2) 2000-1

CONC OF

MGCL2 (

mM

)

-37-

gure 5: E f f e c t o f sodium f l u o r i d e on c y c l i c AMP-dependent protein kinase a c t i v i t y i n coronary a r t e r i e s .

Total

p r o t e i n k i n a s e a c t i v i t y was e s t i m a t e d i n t h e presence o f 2 yM cAMP i n t h e f i n a l Values r e p r e s e n t mean o f 2 s i m i l a r

incubation mixture. experiments.

-38-

-39-

F i g u r e 6: E f f e c t o f T r i t o n X - 1 0 0 c o n c e n t r a t i o n on

particulate

c y c l i c AMP-dependent p r o t e i n kinase a c t i v i t y coronary a r t e r i e s .

Protein

kinase a c t i v i t y

in of

the

p e l l e t was e s t i m a t e d , both i n the presence and absence o f 2 yM cAMP i n the f i n a l as d e s c r i b e d i n Methods. 2 s i m i l a r experiments.

i n c u b a t i o n mixture.,

Values r e p r e s e n t mean o f

-40-

280-i (+)cAMP

0.025 % TRITON X - 1 0 0

-41-

F i g u r e 7: Time course f o r c y c l i c AMP e l e v a t i o n and r e l a x a t i o n o f K C l - c o n t r a c t e d bovine c o r o n a r y a r t e r i e s by 1 uM isoproterenol.

C y c l i c AMP l e v e l s and t e n s i o n were

measured i n t h e same a r t e r i a l i n Methods. experiments.

s t r i p s as d e s c r i b e d

Values r e p r e s e n t means ± SEM f o r 5 Asterisks

i n d i c a t e values s i g n i f i c a n t l y

d i f f e r e n t from t h e c o r r e s p o n d i n g z e r o times (p < 0.05).

controls

-42-

-43-

F i g u r e 8: Time c o u r s e f o r c y c l i c AMP e l e v a t i o n and r e l a x a t i o n o f K C l - c o n t r a c t e d bovine c o r o n a r y a r t e r i e s by 10 yM forskolin.

C y c l i c AMP l e v e l s and t e n s i o n were

measured i n t h e same a r t e r i a l Methods. ments.

s t r i p s as d e s c r i b e d i n

Values r e p r e s e n t means ± SEM f o r 5 e x p e r i A s t e r i s k s i n d i c a t e values

significantly

d i f f e r e n t from t h e c o r r e s p o n d i n g z e r o time (p < 0.05).

controls

-44-

4000n 100

r

3500-

h80

3000-

^ 2500co co P 5?

O

•60 6

5004

< •40 >
r-

O