Influence of tetrahydropapaveroline on adipose tissue metabolism in comparison with that of noradrenaline, theophylline and papaverine

Pharmacological

Research

INFLUENCE IN

Communications,

Vol.

8, No.

OF TETRAHYDROPAPAVEROLINE

COMPARISON

WITH

THAT

6, 1976

525

ON ADIPOSE

OF NORADRENALINE,

TISSUE

METABOLISM

THEOPHYLLINE

AND

PAPAVERINE R.M. Institute

Gaion, of

Dorigo,

Pharmacology,

20 November

Received

P.

M.

Prosdocimi

University 35100 Padua

and

of (Italy)

G.

Padua,

Fassina Largo

E.Meneghetti

1975

SUMMARY The

influence

structurally

of related

biosynthesized was

accumulation

and

vivo

FFA

was

activity,

but

CAMP levels.

dopamine) in

and

the

between

of

the

hormone.

synthesis

alkaloid

and,

Papaverine

is

either

noradrenaline. THP has

these

basal

conditions

From

these

results

inhibitory

phosphodiesterase,

but

influenced

by

does

or

effect this

on

enzyme

with

THP lipolytic increasing

not

alter

the

but

potentiates rate,

on

the

made

in

higher

effective

in

an

and

a still

not

CAMP

No significant

experiments, at

be

tissue of

was

effective

itself

can

level

a comparison

more

alkaloid

adipose

the

papaverine.

was by

in

rat

noradrenaline

alkaloid

content

at

(an

that

on

vitro

and

Theophylline

nucleotide

that

from

release,

found

(THP)

catecholaminesand

theophylline

difference

that

the

investigated

noradrenline,

the

to

in

metabolism

action

tetrahydropapaveroline

in

cyclic the

be

of

presence was

adipose

could

that AMP

hypothesis an

the

of put

forth

tissue

different

from

papaverine.

INTRODUCTION Tetrahydropapaveroline alkaloid papaverine

present and

(THP) in

morphine

Papaverum (Shamma,

is

a natural

somniferum 1972).

isoquinoline as

a precursor

of

2

526

Pharmacological

The

biological

compound on

and

was

animal

synthetized liver

Holtz

during

by

the

Holtz

-acetaldehyde, amine.

This

reaction

(1970)

using

liver

that

might

THP

central the

man

Cohen

during

alcohol transmitters

Cohen,

1973b;

labeled

THP

in

of

Davis

1974).

After

and

hypothesis

Walsh,

al.

et

the

Besides

the

of

1970a;

pharmacological

properties

Laidlaw

The

(1910).

effects

activity

1910;

1964;

Kukovetz,

1967;

Fassina

1967a; et

al,,

al.

al.

(1964) in

the

basis

of

tetrahydrobe

biosynthesized

as

false

the

periphery

in

1973a; of

alkaloids

The

contemporaneous

ethanol,

and

the

et

injection

formation

may

be

alcoholism

and

related

its of

primary THP led

involved

in drug

the dependence

1973).

physiological

role

too

investigated

were was

and Holtz

shown

to

of

have

THP,

et

al., and

Simon

et

its first

specific by

both

a papaverine-like

Kukovetz 1967;

Walsh

et

Cohen,

alkaloid

alkaloid

p-sympathomimetic (Laidlaw,

that

Walsh,

possible

in

intravenous

1970a).

is

with

morphine-like

(1970b)

that

al.,

and 1970;

augmented

effects

function

radioactive

by

of

THP,can

brain

Collins,

rat,

et

the

It

biogenic

on the

a group

can

and

acetaldehyde,

pharmacologic (Davis

the

(Davis

metabolite the

(Cohen Rahwan,

formed

finding

in

and

b).

transmitter

which

intake

be

(Holtz

the

Santi

investigated

among

can

1974

vitro

of

that

guinea-pig

incubated

a chemical

later

with

of

in

homogenates

showed

alkaloids,

conditions

were

was

THP

al,,

metabolite shown

as

that

3,4-dihydroxyphenyl-

hypothesis

a role

6,

studies

oxidase

et

with

further

early

system

Holtz

direct

brain

The

findings.

adrenergic these

and

monoamine

dopamine the

dopamine

8, No.

this

previous

s h owed

of

1964a;

was

play

-isoquinoline in

is

of

(1963)

of

al,, of

NAD.

nervous new

et

which

and

al.

Vol.

of

results

incubation

condensation

dopamine

the

et

Communications,

interest

preparations

1963;

al,,

formed

to

by

mitochondrial

et

physiological

evidentiated

tissues,

Research

spasmolitic

19 64b;

Santi

et

Pdch,

1967b;

Santi

al.,

1971).

However

al,, et

al., two

1976

Pharmacological

main

Research

Communications,

differences

must

oxidative

phosphorylation

of

muscle

smooth

rapid

phase

the

of

The

lipid

the

optical

and

mobilizing

and

investigated

in

and

Sheppard

and

this

Institute

done

to

of

vitro

the

evidentiate

In

with

this

but

paper

also

on

the

and

theophylline of

carried

Free

fatty to

buffer,

by

Lee

studies al.,

et

1967)

out

were

level

also

and

in

adipose

on

TGL

adipose

tissue

will

of

noradrenaline,

action to

in

of

only

the

(1974)

relationship a cellular

as

al.

carried

THP not

SO

THP in

their tissue.

hydrolysis be

evidentiate

the

specific

intracellular

districts

determination

of

metabolism.

male

and

indicated

the

were

50

glycerol

1.90 2.5

were

of

Krebs-Ringer

dissolved

in

incubated

for

a further

11 min

TCA

(0.15

and

noradrenaline

reaction ml/sample).

weighed

where

incubation

(each

the

(1955).

bicarbonate

a preliminary

added

period,

1971).

Korn

pooled,

albumin,

30 min,

% ice-cold

37"

to

rats, ml

al.,

medium

according

% bovine After

et

incubation

for

incubation of

into

lipolysis,

(Maragno

the

Wistar

papaverine, at

samples

in

albino

containing

shaker

theophylline

described

and

placed

THP or

a metabolic

and

titrated

(1956)

from

pH 7.2,

addition

recently

receptor

the

previously

were

Dole

-+ 5 mg)

of

with

of

as

acids

pads

end

et

preparation

out

according

the

(Fassina

rat

tissue

tissue

were

and

Some

papaverine,

action

was

activity

AND METHODS

Adipose

Fat

in

on

stimulatory

tissues

of

compared

adipose

MATERIALS

alkaloid

13-adrenergic

the

1967).

(1974).

influence

and

regulating

(200

the

phase

also

effect

cyclase

at

tonic

inhibits

structure-activity

CAMP levels

considered

mechanism

past

decreases

the

no

al.,

derivatives

interaction

has

different

the

tetrahydroq.uinoline

et

the

in

THP

adenyl

Burghardt

in

only

and

(Santi

52;'

1976

: papaverine

whilst

function

6,

inhibits

contraction

isomers

8, No.

evidentiated

contraction,

of

mitochondrial

be

Vol.

50

was

in

and/or pl

of at

0,9 37".

stopped

% NaCl) At

the

by

the

Pharmacological

528 Isolation

and

were

then

for

20 min

assay

at

10,000

100 sample

Cyclic

BaSO

The

4 and

BaSO

10,000

One

columns the

ml

The

lyophilized

water

and

of

this

solution. cyclic

of

eluted

AMP,

to

a

l/5

Kuo

by

and

Cyclic

water. column

columns

assayed

samples

in

was

AG HOW-x8

appeared 2 ml

of

lyophilized

300

mg/ml ml

of

water). distilled

amounts

a very

assayed

to

AMP

different

having

(1970,

eluate;

were

in

ion

added

(0.343

dissolved

both

Greengard

with

the

of

(30min

purified

were

of

ml

centrifugation

solution

nucleotide

supernatants,

supernatant

the

AMP was

the

added

Before

samples,

by

to

histone

those

the

further

ml

was

cyclic In

the

from

ml

AMP.

(1968).

removed

sixth

residue the

to

according

AMP fraction 0.5

the

neutralized al.

added

AMP was

the

centrifuged

cyclic

AMP were

et

was

cyclic

to

of

the

Krishna

and

and

represented

cyclic

from

were

cm)

with

column

purified

aliquots

third

cyc.2i.c

high

of

concentration

directly

in

the

eluate.

Assay

for

according -al.

and

samples

recovery.

precipitate

4

(0.5x5

together

of

determine

Ba(OH)2

TRIS

supernatant

of

Voi. 8, No. 6, 1976

homogenized

purification

chromatography

1972).

the

The

the

of

The

1 M ice-cold

pmoles

method

g)

exchange

from

to

AMP was

5 % ZnS04

at

g. for

centrifugation, control

AMP.

with

material

the

cyclic

neutralized

starting

by

of

Research Communications,

cyclic

AMP.

to

method

(1970)

1972a; &.,

the as

The

described

Fassina

et

al.,

nucleotide

levels

of

Kuo

and

in

our

previous

197213;

were

Greengard

Dorigo

(1970)

papers et

measured and

(Fassina

al.,

1973a;

Dorigo

slope

of

the

curve

kinase

to

the

Kuo

et

et

al., -et

197313). In

our

regarding

experimental

conditions,

the

of

activity

concentration pmoles

of

and

the

Overall

recovery

samples

was

the

the protein

cyclic

AMP was

apparent

Km value

between

of

synthetic 60 and

75

constant, for

between

cyclic

0.5

AMP was

cyclic

AMP added

%. The

experimental

to

and 1.2x10

the

control

data

has

10 -8M .

Pharmacological been

Research Communications,

corrected

for

recovery.

DEAE-cellulose

Materials. histone

(Type

were

the

II)

purchased

(medium

Centre,

Amersham. grade

as

analytical

hydrogen

form,

was

washed

a 50

% (v/v)

at

4"

as

Noradrenaline

was

from

synthetized and

Padua, The always

from

BioRad

papaverine

of

by

(200-400

water

Student

It'

the

and

water.

from

Recordati,

from

the

mesh) in

Pharmaceutical

of

the

hydrochloride

was

significance

calculated

resin

Tetrahydropapaveroline

hydrochloride

statistical

from

distilled

was

Institute

V)

Laboratories

in

monohydrate

the

(Fraction

distilled

suspension

C.Erba. in

AG 5OW-x8

repeatedlyin

bitartrate

theophylline

m-equiv./g)

AMP and bovine albumin .v 32 P-ATP was obtained Sigma. ,l

purchased

kept

0.85

mesh,

cyclic from

Radiochemical

529

Vol. 8, No. 6, 1976

Chemistry, Hoffmann

experimental

La

Roche.

values

was

test.

RESULTS Comparison

between

and

THP on

cyclic

rat

adipose

parallel

of

two

probably

drugs

of

lower,

a small

of

presence

of et

stimulation

able

that

5x10m4M

the

level

found

were

of

used,

(about theophylline

The

were conditions

nucleotide

CAMP basal

increase

in

different

these

alter

when

and

Two

in

in

measured

3 mM theophylline

basal

significant

AMP accumulation

THP was

and

to

fact,

theophylline

theophylline.

1 shows

noradrenaline

al.,

of

and

high In

but

Cyclic

5x10v5M

not

its

experiments.

content,

during

this

levels

are

2 fold)

both

can

be

in

found

1972a).

Nevertheless

is

and

Figure are

noradrenaline,

presence

drugs,

too.

because

series

(Fassina

the

tested

last

the

10 -3 M noradrenaline

of

parallely

the

in

of level.

noradrenaline

concentrations

the

influence

AMP basal

tissue with

actions

the

in which

these

conditions

increases

when

THP induces the

concentration

a significant of

the

drug

higher. These

data

indicate

a possible

action

of

the

alkaloid

both

530

Pharmacological

Research Communications,

Vol. 8, No. 6, 1976

EFFECT OF NORADRENALINE ,THEOPHYLLINE AND THP ON CAMP BASAL LEVELS IN RAT ADIPOSE

TISSUE

I

NE 10%

THEOFt 3X10-3b.3

THP 5X10%

THP 5xVT4M

of noradrenaline, theophylline and THP FIG. 1. Effect on CAMP basal levels in rat adipose tissue. Rat epididymal fat (ZOO -+ 5 mg) was incubated in 2 ml of Krebs-Ringer bicarbonate containing bovine albumin and, where indicated, tetrahydropapaveroline. Noradrenaline or theophylline were then added. After 11 min of incubation, the reaction was stopped and cyclic AMP was extracted and purified as described in the Methods. Reaction mixtures for the assay of cyclic AMP contained, in the final volume of 0.2 ml, 10 umoles sodium acetate buffer pH 6.0, 2 umoles magnesium acetate; 40 pg histone; lo-70 ~1 of tissue and medium extracts; 8 ug of heart protein 1 mumole of $2P-ATP, containing about kinase; counts/min. 1.8~10"~ Incubation and the following steps were described by Kuo and Greengard (1970). The data represent the mean (2 S.E.) of six to eight determinations.

at

the

level

of

adenyl

cyclase

system

or

on

THP

FFA

phosphodiesterase

activity. Influence

of

tissue. were

noradrenaline

Different tested

between represents

the

in

concentrations order

lipolytic the

and

absolute

to

of

evidentiate

action increase

of

on

from

noradrenaline any

the

release

two

induced

and

possible drugs. by

of

THP

difference Figure

the

adipose

two

2 drugs

on

Pharmacological

Research Communications, There

FFA mobilization. the

two

is

no

531

Vol. 8, No. 6, 1976 significant

difference

between

curves. EFFECT OF DIFFERENT CONCENTRATIONS OF NORADRENALINE AND THP ON FFA RELEASE FROM ADIPOSE TISSUE

of different concentrations of FIG. 2. Effect noradrenaline and THP on FFA release from adipose tissue. Rat epididymal fat (100 + 5 mg) was preincubated in 2 ml Krebs-Ringer bicarbonate pH containing 2.5 % bovine albumin and THP, 7.2, where indicated,at 37” for 30 min in a metabolic shaker. Noradrenaline was then added and the incubation continued for 15 0 min.' FFA increases (uEq/g fresh tissue) from control are reported. Each value represents the mean (2 S,E.) of three to six assays.

Cyclic

AMP levels

in

with

THP

and

does

not

alter

with

THP or

with

both

of

alkaloid

induces

the a 2.9

with

the

presence

of

CAMP accumulation, noradrenaline

fold

increase

but it

of

associated

3 mM theophylline

noradrenaline.

and

theophylline

the over

when

it

potentiates

hormone the

(Fig. value

by is

associated

the

action

3). found

itself

In in

fact the

it

Pharmacological

532 presence when

of 5x10 -4 M THP

5x10

theophylline

on

However by

THP

the in

-5 M THP only. is

the

This

present,

The

noradrenaline

maximal

of

Communications,

increase

is

is

by

theophylline

2.4

induced much

induced

Vol. 8, No. 6, 1976

about

potentiation

action

stimulation

presence

Research

is

higher

fold

by (x18).

noradrenaline

and

similar.

Effect of Theophyllme on CAMP synthex stimulated by THP and Noradrenalme

7. L.-l -

x;.9 -

- -+* ,I L----F? 111 lb-bI~

-

THP SX~O-~M

-

THP SXIO-~M

NE 1o-s i-4

* P
FIG. 3. Effect of theophylline on CAMP synthesis stimulated by THP and noradrenaline. Rat epididymal fat (200 _+ 5 mg) was added to 2 ml of Krebs-Ringer bicarbonate buffer containing 2.5 % bovine albumin and THP, where indicated. After preincubation for 30 min at 37" in a metabolic shaker, theophylline and noradrenaline were added and the incubation continued for 11 min. The other experimental conditinns were the same as described in Fig. 1. The data are the means (2 S.E.) of four to six assays. Lack

of

papaverine

conditions

and

typical

1971) in

basal

influence under

inhibitor did

not

noradrenaline of

show

conditions

on

CAMP

synthesis

stimulation.

phosphodiesterase any

effect

or

in

on the

in

Papaverine,

activity

(Beavo

CAMP synthesis presence

basal

of

either 10 -5,

a et

al.,

Pharmacological

Research Communications,

noradrenaline.

The

concentrations

of

results

are

(Fain

and

in

that

is

not

connected

in

adipose

obtained

the

drug

agreement

Rosemberg,

showed

the

are with

1972;

influence any

three

reported

in

the

different Tab.

findings

Fain, of

with

with

1973;

the

of Allen

alkaloid

alteration

other et

on

of

1 and

the

2.

These

Authors

al.,

1973)

who

phosphodiesterase nucleotide

content

tissue.

TABLE

Drugs

data

533

Vol. 8, No. 6, 1976

1.

in

Lack

of

papaverine

effect

on

CAMP basal

CAMP pmoles/g fresh tissue

the incubation medium M cont.

levels

Significance

1733.492250

-22

Papaverine

2x10 -5

1473.622183.94

n-s.

Papaverine

2x10 -4 -3 2x10

1078.142380.44

noso

Papaverine

Experimental value represents significant.

1655.94+ conditions the mean

57.50

n.s.

are described (2 S.E.) of

four

under Fig. assays.

THP had

1. n.s.

Each = not

DISCUSSION The

F-sympathomimetic

activity

of

demonstrated

by

Authors,

both

(Holtz

et

Fassina

in

et present

vitro

is

increase

1967;

the

and two

Santi

et

al.,

1964;

Simon

et

al.,

i971)-

experiments stimulated is

noradrenaline

activity for

i964b;

al.,

The

This by

al.,

different

the drugs.

show by

that

different

different

and

could

that

for

the

Santi

et

p -receptor

in al.,

tissue

the are

vitro 1967;

lipolysis of

from both

already

and

concentrations

significantly

affinity

vivo

adipose

not

suggest

in

been

that

THP. induced

intrinsic very

similar

Pharmacological

534

TABLE

2.

Drugs

in

Lack presence

of

the medium

incubation

M

papaverine of noradrenaline

10 10 2x10

-5 -5 -5

Noradrenaline + Papaverine

10 2x10

-5

Nor-adrenaline +

10

Papaverine

2x10

Experimental value represents significant.

A

the of

-5 -3

result

the

presence

-4

conditions the mean

different

testing

ascorbic

This

discrepancy

acid,

which

was

shown

phosphodiesterase drugs of

1042.13

i

92.24

nos.

1300.35

2

186.41

n.s.

1705.92

2

76.10

n-s.

1572.69

2

109.32

n.s.

described S.E.) of

in

inhibit

in

different

the

other

et

lower

1. n-s.

delay

than

adipose

Each = not

a1.(1967) and

the

THP

the

oxidation

the

maximal

that

of

fact

that

when in

the of effect

noradrenaline. ascorbic

tissue

19701,

al.,

et

activity,

depending

ways,

Fassina

to

the

to

under Fig. assays,

noradrenaline

order

explainedby

Significance

four

by

be

(Hynie

the

250.22

was

could

in

2

of

conditions

levels

1733.49

obtained

acid,

CAMP

Vol. 8, No. 6, 1976

pmoles/g tissue

Regarding

those

on

Communications,

CAMP fresh

activity

catechol-drugs. in

are (2

was

lipolytic

of

THP

effect

conco

Noradrenaline Noradrenaline + Papaverine

Research

may

on

their

is

not

that

a

interact

with

other

specific

mechanism

action. On

levels

in

exists

between

hormone

When

hand

the

and adenyl

same CAMP by

the cyclase

noradrenaline way

as

THP,

accumulation

so

and

FFA

able poor release

to

increase

CAMP

correlation induced

by

alkaloid. is

stimulated

by

noradrenaline

the

the

Pharmacological

Research

intracellular

Communications,

response but

synthesis, with

a great

this

way

is

is

not

widely

THP

is

for

nor-adrenaline,

is

present,

of

a high the

6,

lipolytic

at

during

the

final

increased,

and

the

ratio.

the

level

following

On the is

this

quite

CAMP steps In

contrary

when

the

as

levels

case

of

response.

nucleotide

in

the

lipolytic

response

also

535

1976

appreciable

FFA/cAMP

but

significantly

8, No.

amplified

enhancement

there

Vol.

same are

FFA/cAMP

ratio

is

lower. This to

discrepancy

assume

the

that

THP

adipocyte,

However

et

The

results

the

different

In

1973)

influence

that

by

activity

that

of

there

at

not

stimulatory tissue

lipolysis

(F ain,

1973;

effect

of is

(Beavo

was

as et

al.,

conditions also

lipolysis

et

reported (Fassina

is

true,

it

result

does under

to

have et

brain

in

enzyme

al.,

alkaloid

accumulation.

much to

more

this on

we must

assume

different also

assuming

on

CAMP levels

of

phosphodiesterase

to

be

that

from

suggested

for

than

explain

effect

then

was

again

CAMP

quantitatively

theophylline

al.,

on

an inhibitory

that

that

the and

adipose

inhibition

1973). shown

a phosphodiesterase

or

us

action

the

hypothesis

were

was

1971),

using

of

drugs

evidence

the

Allen

when

nor-adrenaline

However, concrete

of

led

evidentiated

two

has

level

which

theophylline

basal

this

drugs

phosphodiesterase.

association

the

this

yet

Papaverine,

the

itself

theophylline. is

seen

reasonable

If

of

theophylline, of

more

phosphodiesterase. its

was

two

component

level

potentiates

THP

the

another

the

by with

The

of

0

obtained

theophylline

is

have

effect

al.,

FHP activity.

behaviour

at

noradrenaline

fact

fact

could

inhibitory

(Furlanut

of

the

probably

no

and

in

more inhibitor

not

alter

Beavo

in

adipose either

stimulation.

significant

1967;

than

CAMP levels

noradrenaline no

active

influence et

al,,

tissue in

This

drug

Fain

and

on 1971;

536

Pharmacological

Rosemberg, is

1972;

Fain,

qualitatively

THP.

In

of

be

in

other

phosphodiesterase Appleman,

hypothesis

the

could

already

that

that

of

theophylline

that that

have

Klutz

et

al.,

could

be

particularly

found

difference

between

spasmolitic

activity.

Papaverine

was

tonic

of

muscle

inhibits

the

1967)

D The

drugs

for

smooth rapid

reason two

the

THP on role

could

by

(1973)

induces

shows

a stimulation

proportional

increase

between

this

formation

result might

Rodighiero Pharmaceutical preparation Grateful

manuscript.

Miss

Dr.

1973)

0 of

papaverine

inhibit

only

instead

THP

affinity

the

(Santi

et

of

two

the

al.

for

adenyl

of

CAMP content.

the

might

influence

exist

during

report

by

alcohol

Kuriyama

treatment

cyclase

in

with

and

in

the

rats

brain,

A possible

with

a

relationship

tetrahydroisoquinoline

alkaloid

authors

are

Gianfranco

grateful

Chiarelbtto

to

Prof.

Giovanni

(Institute

University

of

Padua)

of for

the

tetrahydropapaveroline.

thanks Nora

al.,

too

brain

ethanol

of

Chemistry, of

and

exist.

The and

the

a recent

chronic

and

Acknowledgements.

to

that

to

connection in

fact

adipose

and

we obtained

alkaloid In

papaverine

phosphodiesterase.

a new

the

states.

of

a

because

but

be the

evidence

CAMP metabolism,

intoxication

and

the

et

THP

shown

of

of

contraction

probably kinds

then

played

Israel

this

on

(Thompson

contraction,

of

different

Considering of

phase

in

interesting

mentioned

phase

as

Appleman

1972;

acts

kinds

been

behaviour

by

case,

8, No.

and

THP

different

Vol.

this

inhibited

be the

tissues,

activity 1971;

clear

from

This

Communications,

is

possibility

different

like

This

the

speculated,

tissue,

It from

this,

phosphodiesterase can

1973).

different

view

Research

to

Mr.

Loughnane

F. for

Daniel

for

correcting

technical the

assistance, English

in

the

6,

1976

Pharmacological

Research Communications,

Vol. 8, No. 6, 1976

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