Mechanism of myocardial protective action of dilazep during ischaemia and reperfusion

Pharmacological

Research

MECHANISM

Communications,

Vol. 19, No. 5, 1987

341

OF MYOCARDIAL PROTECTIVE ACTION OF DILAZEP

DURING ISCHAEMIA

AND

REPERFUSION.

A.

Cargnoni,

E.

Condorelli,

C. Ceconi,

S. Curello,

A. Albertini*

and R.

Ferrari.

University Brescia,

of

Brescia,

Chair

of

Cardiology

and

*Chair

of

Chemistry,

Italy.

SUMMARY The aim of this study was to investigate if dilazep is able to reduce with a direct protective action on the myocardium the deleterious effects caused by ischaemia and reperfusion. For this purpose we used an isolated rabbit heart preparation. The hearts were either perfused aerobically or made totally ischaemic for 60 min (by abolishing coronary flow) or made ischaemic for 60 min and then reperfused for 30 min. Ischaemic and reperfusion damage was measured in terms of alteration in mechanical function, lactate and CPK release, mitochondrial function and tissue content of Adenosine Triphosphate (ATP), Creatine Phosphate (CP) and calcium. Dilazep (10e5 M) was administered in the perfusate either 20 minutes before ischaemia or only during post-ischaemic reperfusion. Ischaemia induced a decline of the endogenous stores of ATP ,and CP, followed by an alteration of calcium homeostasis with increase of diastolic pressure, mitochondria calcium overload and impairment of the oxidative phosphorylating capacities. On reperfusion, tissue and mitochondrial calcium increase the capacity of the mitochondria to use 02 for state III respiration was further impaired and the ATP-generating capacity reduced. Diastolic pressure increased and there was only a small recovery of active tension generation associated with massive CPK release. Administration of dilazep before ischaemia induced a negative inotropic effect which, in turn, resulted in a slowing of the rate of CP and ATP depletion during ischaemia. This protected the hearts against the ischemic, and reperfusion-

0031-6989/87/050341-l

7/503.00/O

0 1987

The Italian

Pharmacological

Society

342

Pharmacological

Research

Communications,

Vol. 19, No. 5, 1987

induced decline in the ATP-generating and 02-utilizing capacities of the In addition, there was a less marked increase in tissue and mitochondria. release were reduced and the recovery mitochondrial Ca++ , CPK and lactate significantly increased. developed pressure on reperfusion was of the Administration of dilazep during reperfusion failed to modify of coronary exacerbation of ischaemic damage caused by the readmission flow. These data suggest that dilazep benefits the ischaemic myocardium via an ATP sparing action.

INTRODUCTION

Dilazep,

a

1,4-diazepin

[3-(3,4,5-

possesses

man (Rau et mainly

1,4-bis

al.,

1968;

trimethoxybenzoyloxy)propil]

coronary

vasodilator

Lemke et

al.,

adenosine-mediated

(Mustafa

1972).

1979;

perhydro-

action

both

This

action

Fujita

in animal

and

in

is assumed to be

et al.,

1980;

Saito

et

al.,

19821.

It has been reported ischaemic

necrosis

moderately al.,

Fujii

of

dilazep

effect terms

of

changes

action

These

an alternative

of the

vascular

latter and/or

the

myocardial

collateral

inducing

"coronary

Chiariello

et

al.,

myocardium

is

also

action

adenosine,

of

blood steal"

1983).

tissue

from

flow

to the

(Marshall Thus,

usually

et

protective

explained

causing

in

favourable

resistance. has been suggested

and exhibits

adenosine-mediated

1975).

1981;

on ischaemic

recently,

can preserve

positively without

et al.,

in coronary

anaestetic its

areas

a potentiation

However,

dilazep

influencing

ischaemic

1974;

that

additional

dilazep

calcium-antagonistic

mode of effects

that

action

are of

(Tamura particular

role

in the

possesses

properties et al.,

1974;

interest

as they

protection

a local

unrelated Tamura

exterted

could

et

to al., play

by dilazep

Pharmacological

on the

Research

ischaemic The

dilazep

aim is

for

the

was during

present

to

reduce

reperfusion

evaluation

of the

since

investigate

isolated,

heart

direct

influences

the the

post-ischaemic

effects

isolated

hearts

is

of agents

either

valuable

or interventions

on the

The use of offers

on coronary

flow.

before

by

rabbit

particularly

study,

in this

with

caused

perfused

are eliminated.

of dilazep

treatment

effects

Langendorff

preparation

effects

if

deletereous

as has been utilized

of eliminating to

was to

some of the

peripheral

ischaemia,

delivered

study

in the

an isolated

no-flow

advantage

the

The use of

myocardium, of

of

and

hearts.

343

Vol. 19. No. 5. 1987

myocardium.

able

ischaemia

Communications,

ischaemia

a model also

the

Dilazep or

after,

reperfusion.

MATERIAL AND METHODS

Perfusion sequence: adult, male, New Zealand white rabbits (2.5-3.0 Kg body -maintained on a standard diet were used. When required, each rabbit was stunned with a blow on the head. The heart was rapidly excised and perfused according to the non-recirculating Langendorff technique, using modified Krebs-Henseleit buffer equilibrated with 95% oxygen, 5% carbon dioxide and 11.0 mM glucose as previously described (Nayler et al., 1980; Ferrari et al., 1986). The buffer solution was derivered to the aortic cannula at 37°C and at a perfusion pressure of 60 to 80 mmHg, maintained with a Watson Marlow rotary pump (HRE MK3). The perfusion pressure was monitored at the head of the aortic inflow cannula with a Statham P 23 transducer or a pressure manometer. This provided a constant flow of 25 + 1.7 ml/min. The hearts were paced at 180 beats/min using suprathreshold rectangular pulses of 1.0 m.sec. duration'as described (Ferrari et al., 19821. A period of 30 minutes equilibration was allowed before any experimental intervention. Then the hearts were either perfused for 90 minutes under aerobic condition or made totally isch.aemic (abolishing coronary flow) for 60 minutes. Left ventricular wall temperature was monitored by means of a micro-thermistor inserted in the wall of the ventricle and maintained at 36 to 37" C. In order to prevent cooling when the flow was discontinued the temperature of the water jacket surrounding

Pharmacological

344

Research

Communications,

Vol. 19, No. 5, 1987

the heart was increased. To achieve that, the water jacted was connected via a two-way stop cock with a second bath where water was maintained at 65" C. Previous experiments showed that the temperature required in the surrounding water jacket to prevent cooling of the hearts was 65" C. During unrestricted perfusion and during reperfusion, the surrounding water jacket was connected with the circulating water bath of the reservoir (adjusted to 40" Cl. At the onset of ischaemia the water jacket was abruptly connected with the second water bath by means of the two way stop cock. In this way, the wall temperature of hearts could be maintained at 36 + 1" C irrespective of coronary flow. In separate group experiments, the hearts, after 60 minutes of ischaemia, were reperfused for 30 minutes at coronary flow of 25 ml/min (Group Al. Dilazep (10e5 M) was administered to the hearts in the perfusate either 30 minutes before ischaemia (Group 8) or during post-ischaemic reperfusion (Group C). Left ventricular pressure measurements: to obtain an isovolumetrically beating preparation a fluid filled ballon was inserted into the left ventricular cavity via the atrium. The intraventricular ballon was then connected by a fluid-filled polyethylene catheter to a Statham pressure transducer (P 23 D6) for the determination of left ventricular pressure as described (Ferrari et al., 1982). Coronary effluent analysis: coronary effluent was collected in a chilled glass vials and assayed, on the same day for lactate, following the method described by Horost et al. (19591 and for creatine phosphokinase (CPK) following the method described by Oliver (1955). Isolation of the mitochondria: mitochondria were isolated at the end of each perfuzonTy differential centrifugation as previously described (Nayler et al., 1980; Ferrari et al., 1982a). Two different isolation media were used. The mitochondria required for oxygen consumption studies were isolated in the medium described by Sordhal et al. (1973) containing 180 mM KCl, 10 mM EDTA, 0.5% BSA. The mitochondria used for the determination of endogenous calcium and of ATP production were extracted in a medium acontaining 250 mM sucrose, and 5 FM ruthenium red (Peng et al., 1977). Ruthenium red, an inhibitor of mitochondrial calcium uptake, was included to prevent calcium accumulation during the isolation procedure. Oxygen consumption measurements: rates of oxygen consumption were monitored polarographycally at 25" C using a Clark type electrode. 1.25 mg/ml of mitochondrial protein was suspended in 2 ml of a solution containing 250 mM sucrose, 3 mM KH2P03, 0.5 mM EDTA, 3mM glutamate, pH 7.4 adjusted with Tris buffer and allowed to equilibrate for 1 min. State III respiration was initiated by adding 0.5 mM ADP. Protein by the

determination: method of Bradford

Mitochondrial protein using BSA as standard

concentration (Bradford,

was determined 1978).

Pharmacological

Research

Communications,

Vol. 19, No. 5, 1987

345

Mitochodrial ATP production: ATP synthesis was determined in the medium used for oxygenconsumption. Synthesis was initiated by adding 0.5 mM ADP. Two-hundred pl samples were taken before and at 6,15,30,45,60,120 and 180 seconds after adding ADP. They were then mixed with 50 ~1 of 10% perchloric acid on ice. Precipitated protein was separated by centrifugation and the ATP content of the supernatant was determined enzymatically using the method of Lamprecht and Trautschold (1974). The total amount of ATP produced was calculated as the ATP present in the reaction chamber 15 seconds after the transition from state III to state IV respiration. Mitochondrial -calcium absorption spectrometry. 500 ~1 HN03. Lanthanum of 10%. CaC03 was used

calcium content was determined by atomic -content: Mitochondri al pellets were digested overnight in chloride was added to provide a final concentration as a standard

Tissue ATP and creatine phosphate ( cp, : the perfusion of the hearts in --which ATP and CP levels were to bedetermined was termined by freeze clamping the left ventricular apex with aluminium tongs. The frozen muscle was then pulverized, homogenized and assayed for ATP and CP following the method of Lamprecht and Trautschold (1974). at the end -Tissue -calcium: left ventricular muscle were tissue. The extracts were calcium content was assayed obtained by drying samples to

of the perfusion period duplicate samples of taken and digested in 1 ml HN03 per g weight diluted in 25 ml of deionized water and the as described above. Total tissue water was constant weight at 95" C.

Statistical analysis: Results are expressed six experiments. Significance tias calculated P = 0.05 as the limit of significance.

as mean + S.E.M. of by StudeGt's t test,

at least taking

RESULTS

Preliminary there

was

a

mitochondrial

20

slight, function

Figure decline

studies

1 (AI

of

after

increased

to

not

the

and tissue

31 + 3.4

the

pressure. onset

that

significant

shows that

developed

min

showed

of

mmHg.

content abolition Resting

ischaemia Reperfusion

after

90 min decline

of of

aerobic

perfusion

developed

pressure,

of ATP and CP. of coronary pressure

began

and by the resulted

flow to

end of

induced

rise 60

in a rapid

a rapid

progressively min

it

had

and sustained

346

Pharmacological

further

increase

in resting

Dilazep

pressure. experimental

was used

model,

significantly before

0.01)

reduced

(FIG.

1,B).

rise

pressure

recovered

dilazep

administration.

failed

to FIG.

in

this the

but

pressure

group pressure

that (B)

recovery

to

to

occurs

during

the (P <

reperfusion

percentage

of

by the

systolic

hearts

during

hearts

of

abolish,

significantly

developed

of the

our

Administration

and it

only

in

required

not

the

was given

developed

10m5 M because,

1,Bl.

delay,

pressure

mechanical

2 shows that of the

of

CPK into abolished

the

of

rate

reperfusion,

during

previously

ischaemia

before

reperfusion,

it

(compare

FIG.

l,C

the

reperfusion

of the

hearts

there

was

accumulated coronary

completely

CPK

leakage

dilazep

did

lactate

effluent.

the

not

modify

and a marked

Administration

lactate from

control

release

and

myocardium. the

of

and substained dilazep

before

significantly

When

added

reperfusion-induced

reduced only

during

release

of

or CPK. Table

I shows that

oxidative hearts rabbits unchanged further

to

When dilazep the

(FIG.

of

1,Al.

a washout

lactate

minimum

pressure

in resting

was 88% of

improve

Figure

release

concentration

the

Vol. 19, No. 5, 1987

25% recovery

was

in resting

Furthermore,

only

of

was able

increase the

with

Communications,

at a concentration

developed

ischaemia

ischaemic-induced

with

this

reduce

dilazep

pressure

Research

administration

phosphorylating after

90

(Group but decline

activity

min of A)

RCI, of

were

aeroic

of dilazep of the

QO2 and ADP/O were these

mitochondria

perfusion.

made ischaemic,

indices

of

made no difference

When the the

yeld

reduced. mitochondrial

isolated hearts of

to the from

from

the

control

mitochondria

Reperfusion function

resulted and

was in a in

a

Pharmacological

Reseerch

Communications,

Vol. 19, No. 5, 1987

tAEROBIA+TOTAL

ISCHAEMIA

347

-+REPERFUSlONj

100 L V. PRESSURE mm Hg meant S.E

60 60 40 20 1 1001

r---T+-

-60

FIG.

1 Effect

of dilazep

of the mean

isolated +

the

f ow

during

of

mitochondrial

of

ischaemia,

20

’

1

1

’

’

’

1

30

40

50

60

70

60

90

ventricular

developed

rabbit of

mean all for

restored Group

hearts.

the

were

abolished

was

A = Control;

Dilazep

hearts

was

it

mitochondria

minutes

error

The

reperfusion

10

on left

standard

coronary

Group

0

and perfused

experiments

decrease

-30

to the

B = Dilazep

paced 60

and resting

Results

of

MINUTES

at

pressure

are expressed

least

six

as

separate

(see

text).

At

minutes

and

then,

during

25

ml/min.

control before

values

of

ischaemia;

time

Group

0

C

=

reperfusion.

from

yeld. control

Furthermore, hearts

and reperfusion

from

figure

3 it

appears

gained

some calcium

during

caused

a much larger

increase.

the

that 60 This,

348

Pharmacological

10

0

20

Research

Communications,

0

30

Vol. 19, No. 5, 1987

lo

20

30 MINUTES

o--o .

FIG.

2 Effect

of

effluent

dilazep of

expressed

turn,

into

addition

phosphorylation

However, the

I and FIG.

to the

signif

icance

perfus

ion

aerobic

See Fig.

the

made activity

no or

mean

to

of

six

further

between

obtained

during

explanation.

phosphorylation

of

are

separate

difference

hearts

amount

coronary

Results

those

reperfused-control total

the

hearts.

and

1 for

in

of the

of oxidative

and the

of dilazep

as

the

converted

ATP produced

ADP

in response

reduced. the

hearts

difference

perfused

to

the

ATP-producing

for

90 minutes

mitochondrial

capacity

or

under

oxidative

calcium

content

31.

administration

mitochondria

of the

of ADP was significantly

condition

(TABLE

error

from

Administration aerobic

-t standard

in an impairement

slowly

release

rabbit

during

isolated

and CPK

perfused

and reperfusion.

ATP relatively

to the

mean

obtained

resulted

mitochondria

lactate and

P relates

ischaemia

in

on

isolated

as

experiments. values

CONTROL IGROUP A) DlLAZEP i10-5M1 BEFORE ISCHAEMIA IGROUP SI DILAZEP ho-5 k# DURING REPERFUSION (GROUP cl pc 0.05 . . pco.01

against

of dilazep the

before

deterioration

the

onset

in function

of

ischaemia caused

protected of

ischaemia

Pharmacological

Research

Communications,

DILAZEP

CONTROL

BEFORE

q

l

FIG.

3 Effect ATP

of dilazep

agent

and

ruthenium

red to ensure extraction.

mean

least

of

the

untreated and reperfusion under

maintenance

and the (TABLE these

I)

six

and it

pathological

of mitochondrial

DILAZEP

&8

DURING

lD-5 M

REPERFUSION

REPERFUSION

calcium were

the

content

isolated

presence they

of

separate

in the

their

between

conditions ATP production

P relates

value

of

a

amounts

endogenous

experiments.

the

absence

as mean -t standard

dilazep-treated reduced

and mitochondrial

micromolar

retained

are expressed

difference

relative

M

. . pr 0.01

that

Results

of at

significance

in

lo-5

ISCHAEMIA

ISCHAEMIA

The mitochodria

chelating

the

the

pso.05

on mitochondrial

production.

during

occurring

q

AEROSIA

349

Vol. 19. No. 5, 1987

obtained

of

calcium error

of

to

the

for

the

series. gain

in mitochondrial

(FIG. even

3). after

calcium

This

resulted

in

60

minutes

of

350

Pharmacological

ischaemia added 15

and another

directly

t.~Fl did

to the not

mitochondria figure during

3 it

30 minutes

of reperfusion

incubation

medium

alteres

the

converted is

also

Research

ADP to

evident

post-ischaemic

rate

that

reperfusion,

to

at which

(FIG.

provide the

ATP (results

3).

a final

ischaemic not

when dilazep it

Communications,

shown).

to

When dilazep

was

concentration

of

and/or

was given

failed

Vol. 19. No. 5, 1987

reperfused

From table to the

protect

I

hearts

and only

mitochondrial

function.

TABLE I - Effect

of dilazep

on mitochondria

AEROBIC PERFUSION

function.

ISCHAEMIC PERIOD

ISCHAEMICPERIOD

60 MINUTES

(60 MINI + REPERFUSION (30 MINI

TREATMENT

90 MINUTES

YELD GROUP A GROUP B GROUP C

10.9t1.9 11.071.7 ----

9.3t2.6 lO.BTl.l* ----

6.3t1.9 9.2:1.1** 5.373.1

RCI GROUP A GROUP B GROUP C

18.6t1.9 17.3x0.5 -----

9.8t1.2 16.3+1.4** ----

4.4+1.2

22.8+3.2** 5.6T2.3

QO2 GROUP A GROUP B GROUP C

242.7t22.1 242.9z13.4 ------

197.3+19.3 221.9+12.4* -------

68.8t20.1 166.1+26.4**

82.5724.3

ADP/O GROUP A 2.4~0.2 2.OtO.l 1.5to.3 GROUP B 2.420.1 2.5TO.3" 2.370.2** --------GROUP C 1.470.3 *P < 0.05; **p < 0.01 P relates to the significance of the difference between the results obtained from mitochondria from the hearts of control rabbits (GROUP A1 and those obtained from the dilazep series (GROUP B and GROUP Cl. Results are represented as mean t standard RCI = error of the mean of six separate experiments. Respiratory Control Index; QO2 = Oxygen Quotient (n atoms/mg prot/min); ADP/O = Adenosine Diphosphate/Oxygen, Yeld=mg/prot. g. wt weight.

Pharmacological

Research

Figure induced

4

Communications,

shows that

a depletion

gain

in

tissue

effect

on ATP, but,

of

DILAZEP

FIG.

4 Effect are

of dilazep expressed

q

content

separate

experiments.

P

difference

between

otained

dilazep

treated

series.

of

it

10-5 M

q

as mean + standard

values

content

DURINO

calcium of

the

induced

DILAZEP

IBCHAEMIA

ISCHAEMIA . p s 0.05 . . pco.01

on tissue

tissue

a small

dilazep

aerobically a significant

10-5 M

REPERFUBION

. ...’ ..*.a.* ..* *.*. ..*.*.* .::: .......:. a

:a.-.-.. ..:*:j ‘. -:::::: ~~

AEROBIA

ischaemia

Administration

ischaemia,

BEFORE

0

in a large

calcium

before

expected,

of ATP and CP and only

ATP or CP.

CP and tissue

CONTROL

as

resulted

tissue

when given

351

hearts,

stores

Reperfusion

had

hearts,

control

endogenous

calcium.

and in no recovery

perfused

in the

of the

overload no

Vol. 19, No. 5, 1987

of

REPERFUSION

ATP,

error

relates for

of the

CP and ca cium.

Results

mean of

at least

six

signifi

ante

the

to

the

the

untreated

and the

of relative

Pharmacological

352

preservation and in those significant

of the

cardiac

subjected

to

reduction

However, failed

to

tissue

of

stores

tissue

plus

during

gain post

ischaemic

hearts

resulted

of tissue

ischaemic

accumulation

Vol. 19, No. 5. 1987

This,

reperfusion.

reperfusion-induced

calcium

Communications,

of ATP and CP in the

ischaemia

when administered

reduce

Research

in a

calcium.

reperfusion,

or to improve

dilazep

the

recovery

of

ATP and CP.

DISCUSSION

This hearts

study before

function

reduced,

were

developed On the

to reduce coronary

of

During

reduced.

contrary,

exerts

stores

pressure

administration

was better

endogenous

release

the

ischaemia

Mitochondrial

effect

maintained,

overloading

with

there a smaller

of dilazep

of

ischaemic

damage

and

the

isolated

the

myocardium.

with

calcium

was

and

CPK

lactate

was a greater rise

only

to on

preserved

reperfusion,

generation,

exacerbation

a protective

ATP were

administration

the

of dilazep

recovery

of diastolic

during caused

pressure.

reperfusion by the

of

failed

readmission

of

flow.

At should

the

present

protect

ischaemia It

or

subjected

there

isolated

is

heart

no certain muscle

explanation

against

the

as

to

why

deletereous

dilazep

effects

of

a potentiat

ion

and reperfusion. is

of adenosine rate

shows that

not

possible

effect increase to total

to explain

resulting of coronary ischaemia

these

in peripheral flow, was used,

as

resu Its

in term

dilatation, an

in which

isolated temperature,

of

reduction heart

in heart preparation

heart

rate

and

Pharmacological

Reseerch

coronary

perfusion

of

dilazep

the

drug

were

cannot

well

be explained

considering medium

1982;

Colli

et

We believe

that

the

beneficial

the

rate

et

the

fact

dependent

that

on

existing

before

appears

that

dilazep

and

minutes

of

dilazep.

the

from

during

homeostasis, Ferrari

were

to

et

al.,

1985).

the

mitochondria

with

their

functional

survival

et

1982bl

Ferrari

al.,

reenergization

of the

production

It obtained

in

interesting the

calcium-antagonist observation (Nayler

et

al.,

to

isolated

3).

also

This,

transport

notice

such in

Bourdillon

does

hearts

avoided,

pathway,

with

similar

rabbit

hearts and

and Poole-Wilson

with

available

(Nayler

1981;

overloading

et

results

of

ensuring al.,

1982;

in a rapid

restoration

of ATP

activity.

that

1971 was later

60

intracellular

Williams would

as verapamil

after

thereby

in turn,

of

treated

increase,

of contractile here

stores

and

1967;

1 it

administration

and sodium

not

be

hydrolisis

ATP remained

calcium

et al.,

and perfused

Fleckenstein 1980;

should

resumption

agents, from

to

is

From figure

permeability

calcium

(Lehninger

and consequently is

respect

ATP

ATP and CP of

lies

ischaemia

of

the

sufficient

membrane

electron

1974;

we founded

1970).

group

hearts,

tissue

(FIG.

of

al.,

during

rate

by

that

in the

of

calcium

et

which

ATP

and on the

appears

with If

of dilazep

was reduced

maintain

particularly

effect

activity

(Nakajima

in CP and

higher

group

protective

antiaggregatory

and Spierckerman,

4 it

in this

ischaemia

of fall

pressure

ischaemia

Thus,

effect

activity

figure

the

1983).

(Kubler

developed

the

was emploied

al.,

physical

ischaemia

In addition,

controlled.

as an asanguineous

353

Vol. 19. No. 5, 1987

al.,

Valori

in

Communications,

results

have

been

by pretreatment nifedipine.

confirmed 1982;

with

The

by many

first authors

DeJong et

al.,

354

Pharmacological

1982).

Therefore,

it

seems that

calcium-antagonists of

depletion

involve

inotropic

study

but

excitatory

it

calcium

through

When given and CP tissue turn,

only

It

been after

slow

calcium

the

cell

membrane

when

heart

which

myocardium

ischaemia

added

al.,

dilazep

failed calcium

damage

the

in

the

the rate

underlaing

of

et

rather

than

or reperfusion

of

massive

this

influx

of

1974). to

improve

overload, with

become

permeable 1986;

accumulation,

the

ATP

which,

no

flow

in

recovery

emploies on its

on a specific or preservation

happen

and

of

to that

through

larger

the of

molecules

The finding

reduce the

during

abnormalities

1984).

drug

at least

in the

complex

metabolical

negative

inhibition

calcium

induces

failed

that,

of not

to calcium

suggests

and confirms

depending

of

does

Poole-Wilson

reperfusion

effect

entry

ischaemia

readmission

on

protective probably

the

the

et al.,

membrane its

period

but

calcium

preparation

ischaemia

the

investigated

(Tamura

myocardial

that

prolonged

channel,

mitochondrial

the

being

and of slow

The mechanism

mitochondrial

suggested

as CPK (Ferrari

preserve

reperfusion,

further

to

on a reduction

sarcolemma

and to reduce

in

has

dilazep,

the

dilazep

an ability

has not

dependent

of

Vol. 19, No. 5, 1987

function.

reperfusion

such

is

Communications,

effect

ischaemia.

of dilazep

during

content

resulted

mechanical

during

effect

likely,

protective

a common mechanism:

of ATP reserves

negative

the

Research

that

tissue is

not

and able

isolated

heart

changes

inotropic

effect

before

of calcium

influx

during

of membrane

permeability.

to

in

Pharmacological

Research

Communications,

355

Vol. 19, No. 5. 1987

ACKNOWLEDGEMENTS

This

work

86.01986.56.

was supported

We thank

preparing

the

Palmieri

for

Miss

by the Ornella

manuscript their

and

technical

Italian

C.N.R.

de1 Ciello

for

Miss

Cristina

grant

84.02569.56

secretarial Capelli

and

assistance and

Miss

in

Michela

assistance.

REFERENCES

BOURDILLON,

P.D.,

BRADFORD, M.M. CHIARIELLO,

s.,

Pharm.

Res.

BREVETTI,

MADERNA, Corn.,

DE JONG,

J.w.,

Pharmacol.

81,

15,

72,

Circ.

J.

Cardiol.,

P.,

MORAZZONI, G.,

50,

360.

248.

GENOVESE,

Int.

Res.,

A.,

2,

CATAFFO, A.,

AMBROSIO, G.,

339. SPERONI,

C.,

TREMOLI,

E.

(19831,

DE TOMBE, P.P.,

KEIJZER,

E. (19821,

Eur.

J.

89. WILLIAMS,

Metabolism,

DI LISA,

A.J.,

CALDARERA C.M.,

(1982a),

F. (1982bl HARRIS P.

In Advances (Edsl,

Clueb

in

Studies

on

Publication,

p. 245.

R.,

Cardiol.,

14,

737.

FERRARI,

R.,

ALBERTINI,

VISI~LI,

G.,

(19821,

593.

FERRARI,

R .,

P.A.

Biochem.,

HARMENSEN, E.,

R.,

Bologna

Anal.

M. (19831,

COLLI,

Heart

(19781,

M.,

CONDORELLI,

FERRARI,

POOLE-WILSON,

DI LISA,

0.

(19861,

F.,

A.,

RADDINO, R.,

CURELLO, S.,

J. Mol.

Cell.

VISIOLI,

0.

(1982b1,

CECONI, C.,

Cardiol.,

5, 487.

DI LISA,

J. Mol.

F.,

Cell.

RADDINO,

Pharmacological

356

FLECKENSTEIN, (Eds),

A.

Academic

FUJII,

M.,

A. (19811,

332,

In Calcium

London

ISHIDA, J.

31,

Y.,

H.J.,

68,

KRENTZ,

Heart,

(19711,

Harris

Vol. 19. NO. 5. 1987

P.,

Opie

L.H.

H.,

NAKAMURA,

p. 135.

OKAMATSU, S.,

TOMOIKE,

K.,

MORITOKI,

H.,

OHARA, M.,

TAKEI,

M.

343.

F.H.,

BUCHER, T.

(19591,

(1970).

Mol.

Biochemische

Zeitschrift,

18.

KUBLER, W., SPIECKERMANN, LAMPRECHT, Bergmeyer

W., H.U.

P.C.

TRAUTSHOLD, (Ed),

A.L.,

LEMKE, D.,

BRDCK, N.,

CARAFOLI,

J.

(19741,

RDSSI,

ZECHEL, H.J.

New York

C.S.

Cardiol.

1, 351,

of Enzymatic (19741,

(19671,

(19721,

(19741,

Cell.

In Method

Press Inc.,

E.,

PARRAT J.R.

MARSHALL, R.J.,

E.

Academic

LEHNINGER,

281,

S.,

Communications,

2067.

IZUMI,

Pharmac.,

and the

and New York

W., KOYANAGI,

Arzneim-Forsch,

Br.

HDROST,

Press,

KIKUCHI,

FUJITA,S., (19801,

(19711,

Research

Adv.

p.

Analysis,

2101.

Enzymol.,

Arzeimittel-Forsch.,

Naunyn-Schmiedeberg's

29, 22,

Arch.

259.

639.

Pharmacol.,

427.

MUSTAFA, S.J. NAKAJIMA,

(19791,

K.,

SHIRAI,

Arzneim-Forsch/Drug NAYLER,

Biochem. K.,

FERRRARI,

28,

TAKIMOTO,

Research,

W.G.,

Pharmacol.,

24, R.,

M.,

2617. SAT0 H.F.,

AOKI,

M. (19741,

1899.

WILLIAMS,

A.J.

(19801,

Am. J. Cardiol.,

46,

242. OLIVER, PENG,

T.A.

(19551,

G.F.,

RANE,

Cardiol., RAU, (1968).

Biochem.

J.J.,

J.,

61,

116.

MURPHY, M.L.,

STRAUB, K.D.

(19771,

J. Mol.

Cell.

9, 897. G.,

TAUCHERT, M., Verh.

Deut.

Ges.

BRUCKNER, J.B.,

EBERLEIN,

Kreislaufforsch.,

34,

385.

H.J.,

BRETSCHNEIDER, H.J.

Pharmacological

SAITO,

Research

D.,

KUSACHI,

Forsch/Drug

Res.,

SORDHAL,

L.A.,

Physiol.,

244,

TAMURA,

ISHII,

H., T.,

TAMURA,

Jpn., VALORI, Res.,

71,

Bologna

9,

S.,

357

Vol. 19, No. 5, 1987

NAGASHIMA,

H.,

HARAOKA,

S.

(19821,

Arzneim-

1029.

McCOLLUM,

W.B.,

WOOD,

W.G.,

SCHWARTZ, A. (19731,

Am. J.

497. NAKAMURA, M.,

KIRIHARA, H.,

J.

SHIROSAWA,

WADA, S.,

(19741, Y.,

KUSAJI,

The Clinical

HORI, H.,

T., HAYASHI, H., YAMAUCHI, Y., Report,

KONDO, A.

8,

1354.

(19751,

Folia

Pharmacol.

757.

V.M.,

LEONE, G., TRAISCI G., BIZZI,

B. (19821,

Arzneim-Forsch/

Drug

32, 403.

WILLIAMS, Heart

Communications,

A.J.,

CRIE,

Metabolism, (1982a),

J.S.,

FERRARI, R. (19821,

CALDARERA C.M., p.

173.

HARRIS P.

In Advances in Studies (Eds),

Clueb

Publication,

on