Modulation of lipolytic activity in isolated canine cardiac sarcolemma by isoproterenol and propranolol

Vol. 90, No. 3, 1979 October

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956-962

MODULATION OF LIPOLYTIC ACTIVITY IN ISOLATED CANINE CARDIAC SARCOLEMMA BY ISOPROTERENOL AND PROPRANOLOL Richard

C. Franson,

David

C. Pang,

and William

Department of Biophysics, Medical Virginia Commonwealth University, Received

August

B. Weglicki

College of Virginia Richmond, Virginia

23298

28,1979

Summary: Cardiac sarcolemmal preparations isolated from dog were tested for membrane-associated phospholipase A and lipoprotein lipase activities. The sarcolemma hydrolyzed 1-acyl 214C-linoleoyl 3-glycerophosphorylethanolamine at pH 7.0 to form predominantly 14C-lyso PE with 5 mM EDTA and 14C-free fatty acid with 5 mM Ca2+ suggesting the presence of both phospholipases Al and A2 and/or lysophospholipase activities in these reparations. Sarcolemmal PLA activity was stimulated 300% by 10-5 to lo- g M dl-isoproterenol; this stimulation was blocked by 10m4 M dlpropranolol. Lipoprotein lipase activity associated with the sarcolemmal fraction was enhanced lo-fold by 10m5 M dl-isoproterenol; stimulation was blocked by dl-propranolol. Thus, the activities of membrane-bound lipolytic enzymes appear to be modulated by 8-adrenergic agents in canine cardiac sarcolemma and could affect lipid dependent enzymes and/or membrane permeability. INTRODUCTION Pharmacologic fatty

acids

lead

(1) while

to damage from

the

of hamster

characterized this

report

vities

in heart.

is

phospholipid

little

agonists We have

and canine

by isoproterenol

with

experimental

that

are lipolysis

thought

to be mediated lipolysis

described sarcolemma in

animals

in adipose

on sarcolemmal

enzymes

of free

indistinguishable

concerning

cardiac

the modulation

levels

is known

recently

hydrolyzing

we describe

serum

The increased

and CAMP, but

of 8-adrenergic

isolation

doses

of the myocardium administration

receptors function

elevate

or repeated (2-4).

isoproterenol

effects

membrane

large

infarction

8-adrenergic direct

of isoproterenol

and necrosis

from myocardial tissue

doses

these

of sarcolemmal

by the and

a procedure (5)

and have

systems lipolytic

for

(6).

In

acti-

and propranolol. MATERIALS

AND METHODS

enriched 5 to 8 fold in (Na++&-Mg2+ Sarcolemmal (SL) membranes, ATPase activity, were isolated from canine myocardium as previously described (5). Protein was determined by the method of Lowry (7) using BSA as a standard. Phospholipase A activity (PLA) was measured using lacyl 2-14C-linoleoyl 3-glycerophosphorylethanolamine (PE) substrate (8). Reaction mixtures in a total volume of 1.0 ml contained 25-100 ug SL protein, EDTA or CaC12 as indicated, freshly prepared dl-isoproterenol

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Vol. 90, No. 3, 1979

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100 pmoles Tris-Maleate pH 7.0 and 50 nmoles of 1-acyl 2-14C-linoleoyl Reaction PE (9,000 cpm added as an aqueous ultrasonic suspension). mixtures were incubated for 2 - 4 hours at 37OC and activity was linear Reactions were stopped with time and protein under the conditions used. and the products isolated as previously described (8). PLA activity is expressed as nmoles of product (LPE + FFA) formed/hr or as per cent of control. Lipoprotein lipase (LPL) activity was measured using Intralipid as a stabilizing agent (9). The substrate consisted of 2 vol. of 20% w/v BSA, 1 vol of 0.7 M Tris-HCl buffer 0.5 vol rat serum, 1 vol of water, 0.5 vol Intralipid and sufficient l- 14C-triolein dispersed by sonication to yield 50,000 cpm per incubation. The Intralipid was preincubated with serum for 30 min at 37OC prior to addition of the above components and then sonicated. Reaction mixtures in total volume of 1.0 ml contained 0.5 ml substrate, 25-100 ug sarcolemmal protein, 100 nmoles Tris-HCl, pH 8.1, 0.1% Triton X-100 and the indicated concentrations of freshly prepared dl-isoproterenol and dl-propranolol. Reaction mixtures were incubated for 3 hrs, the reaction was stopped and lipids were extracted and separated as previously described (9). The only radioactive product formed was 1-14C-oleate. Lipoprotein lipase activity is calculated as total cpm FFA divided by the total cpm lipid and All values are corrected for nonis expressed as per cent of control. enzymatic hydrolysis. RESULTS The hydrolysis

of PE by sarcolemmal

and EDTA concentration Ca2+ or EDTA moderate FFA and LPE, are duct sition. major action

is

is

hydrolysis

formed.

of PLAl

and could

In the presence

be formed

and lyso-PLA.

as a function In the

absence

and equal

of Ca2+ of added

quantities

of

of EDTA the predominant

pro-

of a PLA specific of 5 mM calcium,

by the action

Hydrolysis

EDTA mM

Figure

1.

of PE occurs

14C-LPE indicating the action By contrast, in the presence

product

PI&

shown in Figure

for the l-po14 C-FFA is the

of PLA2 or the concerted

of PE with

increasing

protein

COCI,

1 Hydrolysis of PE as a function of Ca*+ or EDTA. Standard as described in Methods. contained 100 ng reaction mixtures. sarcolemmal (SL) Protein, EDTA or CaC12 as-indicated, and were incubated for 2 hrs at 3i'OC. PLA activity is expressed as nmoles product formed/hr; LPE I--- ) refers to lysoglycerophosphoryletbanolamine and FFA (---), 14C-free fatty acid. AI1 values are corrected for non-enzymatic hydrolysis which was less than 3% in all experiments.

957

Vol. 90, No. 3, 1979

BIOCHEMICAL

AND BtOPHYStCAL

RESEARCH COMMUNICATIONS

r 25

100

50

03

10-10

I

I

10-e

10-6

lsoproterenol

(M)

10-4

Figure

2 Hydrolysis of PE as a function of protein concentration. Standard reaction mixtures contained 25-100 pg SL protein, and were incubated for 2 brs at 37OC. PLA activity is expressed as nmoles of FFA or LPE formed/hr (Figure 2A) and as nmoles total product (FFA + LPE) formed/hr (Figure 2B).

Figure

3 Effect of dl-isoproterenol on hydrolysis of PE. Standard reaction mixtures contained 50 pg of SL protein, 5 mM CaC12, the indicated concentrations of dl-isoproterenol and were incubated for 4 hrs at 370C. PLA activity is expressed as percent of control (control = 1.0 nmole product formed/hr= 100%).

(Figure then

2) demonstrates decreases

sequential

as total

a nearly

linear

Since creased

that

lysophospholipid

concomitantly

with

product

formed

relationship

previous

is

reports

endogenous

than

dose-response

(not (10'5M)

shown). As shown with

when hydrolysis pg protein

is (Figure

2)

noted.

phospholipid

Interestingly,

M and less

and

suggesting

that

turnover

&adrenergic in perfused

of dl-isoproterenol

agonists cardiac

in-

tissue

on the endogenous

PLAs

that hydrolysis of PE by the SL fraction dl-isoproterenol in the presence 300% by 10B5 to 10-k

was stimulated Ca2+.

Thus,

initially

of 14C-FFA

(LPE + FFA) per

suggested

00) , we examined the effect of SL. Figure 3 illustrates

accumulates

the appearance

PE * LPE + FFA.

degradation:

expressed

lar

I

1

0

concentrations 10-g

M had little

with in Figure

effect

isoproterenol 4 the

of isoproterenol on enzymatic

was noted

stimulation

EDTA or Ca2+ was totally

in

of

than 10m4 A simi-

activity.

the presence

of EDTA

of PLA by dl-isoproterenol blocked

9.58

greater

by the addition

of 10e4 M

Vol. 90, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL

r

RESEARCH COMMUNICATIONS

Llpoproteln

0’ E 1000

LIpme

B b !s 8 6 500 a

lsoproterenol

lo-5

100

0 4

Isoproterenol(M)

(M) -

~ropranolol

IO -5

-

10-4

10-4

10-4M

05

25

50

100

PROTEIN

(pg)

Figure

4 Hydrolysis of PE as a function of dl-isoproterenol and dlpropranolol. Standard reaction mixtures contained 50 ug SL protein, 5mM Ca*+ or EDTA, the indicated concentration of dl-isoproterenol and/or dl propranolol, and were incubated for 4 hrs at 37OC. PLA activity is expressed as percent of control + % S.D. (control = 1.4 t 0.4.nmoles product formed/hr= 100%).

Figure

5 Effect of isoproterenol on sarcolemmal LPL activity. Standard reaction mixtures, as described in Methods, containing the indicated protein and dl-isoproternol concentrations were incubated for 3 hrs at 37'C. LPL activity is calculated as cpm 14C FFA/cpm total lipid and expressed as percent of control (control = 300 nmoles of oleate formed/hr/mg = 100%).

dl-propranolol

which

homogenates activity shown).

both

(11)

drolysis

and is

we tested

effect

serum dependent

LPL activity

degree

of stimulation

product

formed little

lation protein was less

from

was blocked

by adrenergic

than

10e7M

Figure

(not

cardiac in other

on sarcolemmal

maximally

related

stimulation

organ hy-

5 shows that by 10m5 M isopro-

Interestingly,

to content

of protein.

the The

was almost entirely 14C-FFA l- 14C-trioleate di-glyceride accumulation. Again this stimu-

was evident (not

with agents

of isoproterenol

by 10m5 M dl-propranolol;

no stimulation

the

In

PLAs

(10s4M)

associated

by 10m4M isoproterenol.

glycerol

enhanced

macrophages

substate.

was inversely

or no mono-and

is

was stimulated

and was unaffected

of PE.

by dl-propranolol

(LPL) stimulated

the

(10m5M)

of alveolar blocked

of a 14C-triolein-Intralipid

terenol

with

were

lipoprotein-lipase

fractions

systems

on the hydrolysis

dl-isoproterenol

in homogenates

activities

Since

membrane

had no effect

of PMN leukocytes by 270%;

was 400%;

itself

when

shown).

959

M

10-6~

regardless the concentration

of content

of

of isoproterenol

Vol. 90, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

DISCUSSION Many studies repeated

doses

resembles

have demonstrated of isoproterenol

ischemic

upon early

(2,3).

in this

that

intracellular alterations

norepinephrine

or isoproterenol.

that

rapid

permeability

at the

permeability injury.

of the myometrium suggest

that

mechanism

composition

permeability that

studies

event

resulting membrane

PLA and LPL of canine

sarcolemmal

PMN leukocetes

and macrophages

are

isoproterenol;

the stimulation

is

Why stimulation

pranolol. was not

evident

unclear, doses

membranes

organs

and whole

animal

can stimulate

inositol

phospholipids, as well

however

the effect

should

whole

cells

tissue that

or perfused infer

cortex

organs,

an increased

in adipose

tissue,

metabolism

in heart

is modulated

As suggested

by Christian

tissues

between

lipid. the mechanism

the effects

may be similar.

parotid

(10m4M)

similar

effects data

as well turnover,

with

on phospholipid

particularly glands

cells metabolism

phospholipid turnover whereas our observations

phospholipid

(1%

(19,20);

In the above mentioned

varies studies,

was measured with isolated toward of lipolysis

and triglyceride

agents is poorly underand Lech (22), the simi-

of lipolytic stimulants and inhibitors Thus, CAMP may act as an intracellular

960

isolated

as cholinergic

and submaxillary

by adrenergic et al. (21)

high

made in perfused

and smooth muscle

by which

is

with

was due to

These

adrenergic

(18)

agonist, pro-

activities

expression of enzymic activity Unlike the well-studied control

added

stood.

lipolytic

the inhibition

phospholipid

agonists

exogenously

larity

that

PLAs of

S-adrenergic

of isoproterenol

and species. endogenous

by the

demonstrate

by the g-antagonist,

previously

be an

sarcolemmal

of membrane

and particulate

autoinhibition.

cell

could its

membranes

that

in pineal, of these

with be noted

membranes

membrane

lipolysis

has observed

observations studies

as cerebral

considerably it

and/or

confirm

agonists 17),

(14)

cardiac

can modify

sarcolemmal

and proposed

desensitization

cardiac

of sarcolemmal

Our results

concentrations

Furchgott

of catecholamines

receptor

to isoproterenol

stimulated

of these

at higher

although

in response

systems.

blocked

perfused

concluded

in hyperpolarization

alterations

enzymatic

was

alterations

cell

in deleterious

have

hearts (13)

in catecholamine-induced

the cardiac

focused

(12)

in rat

catecholamine-stimulated

by which

and/or

that

which

peroxidase

et al.

resulting

These

We believe

lipid

alterations

may be an early

important

sarcolemma Marshall

sarcolemma

M).

et al.

of horseradish

of the

or

pathology

have reports

Boutet

diffusion

of large

cardiac

recently

process.

due to permeability

(4 x lo-'

causes

Only

injury

with

ion

administration

to rats

necrosis

events

demonstrated

occur

that

in mes-

in

BIOCHEMICAL

Vol. 90, No. 3, 1979

senger to modulate lipolysis

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

in heart and membrane-associated lipolytic

activities in heart may be modulated by a cascade mechanisminvolving @receptors, CAMP, and phosphorylation. We have previously demonstrated the association of phospholipase A2 activities with cardiac sarcolemma in hamster and dog (6). The generation of 14C-FFA from 1-acyl 2- 14C-linoleoyl PE with increasing time or protein

(Figure 2) indicates

and lysophospholipase activities, sarcolemmal preparations (23).

the combined action of phospholipase Al enzymes previously described in canine This report also demonstrates that

this membranefraction contains lipoprotein lipase activity. cardiac SL may contain an isoproterenol-responsive lipolytic system with broad substrate specificity. bute to the normal metabolism of lipids lipoproteins

(24).

induced cell injury

This enzyme system may contriin cell membranesand circulating

Whether these lipolytic or participate

are important questions for future

Thus, enzyme

enzymes mediate catecholamine-

in ischemic injury

of the sarcolemma

studies.

ACKNOWLEDGEMENTS We thank Mrs. D. Weir for excellent

technical

assistance.

This

work was supported by NIH grants: HL-21116, HL-19148, HL-23142, and HL21493. David C. Pang is a recipient of a Research Career Development Award (HL-00488) from the National Heart, Lung, and Blood Institute. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

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