Alkyl chain homologs of platelet-activating factor and their effects on the mammalian heart

Vol. 161, No. 3, 1989 June 30, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1341-1347

ALRYL CHAIN HOMOLOGS OF PLATELET-ACTIVATING FACTOR AND THEIR EFFECTS ON THE MAMMALIAN HEART Roberto

Levil,

'Department 'Department

Received

Arturo

Genovesel

and R. Neal

of Pharmacology, Cornell New York, NY of Pathology,

May 24,

University 10021

Pinckard* Medical

The University of Texas San Antonio, TX 78284

Health

College Science

Center,

1989

SUMMARY: Platelet-activating factor (PAF; AGEPC) is a potent negative inotropic and coronary-vasoconstricting agent. Minor structural alterations in the PAF molecule are known to greatly affect its biological activity; thus, we have investigated the effects of selected synthetic saturated and unsaturated alkyl chain PAF homologs on the isolated guinea pig heart. The rank order of potency for the negative inotropic effect was C16:0- > Clt3:1- > beef-heart AGEPC > C15:0> C18:0> C14:0-AGEPC; the rank order for the coronary-vasoconstricting effect was C16:0= C18:1= beef-heart AGEPC > C15:0- > C18:0- = C14:0-AGEPC. With the exception of C16:0- and C18:1-AGEPC, the relative potencies for the cardiac and coronary effects of the alkyl chain AGEPC homologs did not correlate well with their relative potencies in stimulating rabbit platelets and human neutrophils. The differences in the rank order of potency for these AGEPC homologs suggest the presence of species and/or target cell PAF receptor heterogeneity. 0 1989 AcademrcPress, Inc.

Platelet-activating choline,

AGEPC)

effects

in

mediator

the

of

anaphylaxis

(4)

arachidonate

this

platelet

first rabbit

basophils

documented

release unsaturated

a

variety

of

alkyl

chain

of

PAF

the

PAF is

(l-3).

and arrhythmias

released (5). do not

a

that

both locally and The cardiac effects depend

antagonized

i.e., and mass

and

stimulated homologs

homologs,

the

existence

autacoid; (C16:0-

analytic that

is

specifically

indicated

phospholipid

acetyl-sn-glycero-3-phosphocholine improved quently, with

phosphoryl-

on the

release

by drugs

known

from

IgE-

of PAF (2,3,6). elucidation

structural

acetylated

laboratories

and

of platelets,

and are

effects

humans

constriction conditions

presence

metabolites

the

sensitized

the

ether

and coronary-vasoconstricting

including

many pathophysiological

require

glyceryl

inotropic

mammals,

cardiac in

The

negative

of various

coronary

of PAF do not to block

potent

acetyl

failure,

systemically

of

has heart

(PAF;

contractile

characterize

of

factor

PAF

of two molecular species 1-O-hexadecyl/octadecyl-2Cl8:0-AGEPC)

spectral

l-O-acyl

analogs

(7).

technology,

inflammatory and

released

cells

several

synthesize

including

PAF analogs,

Subse-

saturated

and

ethanolamineDoo6-291x/89

1341

and and

$1.50

Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

161,

No.

containing enyl the

acetylated

linkages

in from

heart-derived

the

sn-1 position activities

chain

studies PAF

length

(a

it

AGEPC molecule

is

previous

of

AGEPC)

C16:0-,

the

effects on

of

its

selected

of

saturated

and

coronary

knowledge

on

stimulation

has beef-

saturated

C16:0-

or and

PAF relied

alkyl

C18:0-AGEPC.

alterations

activity of

view

alk-l-

semisynthetic

structural

effects In

or

of our

other

synthetic

minor biological

cardiac

contractility

or

even

and

COMMUNICATIONS

l-O-alkyl

employed C18:0-

(9,lO)

affect

on

either

principally

that

RESEARCH

most To date, beyond platelet

(5,8). PAF

of

AGEPC (2-4,6).

AGEPC homologs mammalian

of

findings the

BIOPHYSICAL

having

have

now known

may greatly

beef-heart-derived investigated

that mixture

homologs

Nevertheless,

AND

phosphoglycerides,

pathobiological

originated

Our

BIOCHEMICAL

3, 1989

in

potency on the

(5). use

the

preceding,

we have

and

unsaturated

alkyl

vasculature

function

the of now

chain in

the

heart.

METHODS AND MATERIALS anesthetized Methods: Male Hartley guinea pigs (250-300 g) were lightly Hearts were excised and with CO2 vapors and killed by cervical dislocation. The hearts were perfused at a mounted in a Langendorff apparatus (4). constant pressure of 40 cm of water with Ringer's solution warmed at 37OC and This solution contained (mM): Na+, 160; K+, 5.6; ;;e$librated with oxygen. 2.2; Cl-, 164; HC03-, 5.9; glucose, 5.5; and human serum albumin (HSA; New iork Blood Center), 2.5 mg/ml. Isometric ventricular contractions and bipolar surface electrograms were continuously recorded from the right atrium and left ventricle. Coronary flow rate was continuously monitored by measuring the volume of coronary effluent collected during 2-minute periods. After a period of stabilization (30-45 minutes), single doses of beef-heart AGEPC (lo-l4 to lo-' mol) the C14:0-, C15:0-, C16:0-, C18:0or C18:1-AGEPC or the C16:0-deacetylated derivative lyso~~~~l"p;"o~4'O~~410tps8~1~O~10 mol), dissolved in a constant volume of warm oxygenated Ringer's containing 2.; mg/ml HSA, were injected intra-aortically to determine dose-response relationships. To avoid the possibility of tachyphylaxis, only one dose of AGEPC was administered to each heart. Materials: Beef-heart-derived AGEPC was synthesized and purified as previously described (9); this material was composed principally of C16:0AGEPC, 60-70%; C18:0-AGEPC, lo-20%; with lesser amounts of C14:0-, C15:0- and Cl7:0-AGEPC. Synthetic C16:0- and Cl8:0-AGEPC and 1-O-hexadecyl-sn-glycero3-phosphocholine (lyso-GEPC) were purchased from Bachem Fine Chemicals, CA; C14:0and C15:0-AGEPC were bought from Calbiochem, La Jolla, Torrance, CA; ClS:l-AGEPC [1-0-(10-octadecenyl)-2-acetyl-sn-glycero-3-phosphocholine] was a gift from Dr. Donald Ayer of the Upjohn Co. (Kalamazoo, MI). All phospholipids were analyzed for purity by thin layer chromatography and each of their respective structures verified by fast atom bombardment mass spectra-metry (11) and by gas chromatography/mass spectrometry of the respective 3-heptafluorobutyroyl derivatives (12). All AGEPC alkyl chain homologs were finally dissolved in the lower phase of a mixture of chloroform/methanol/ water, 1:l:O.g and were stored at -2ooc. The concentration of each AGEPC homolog was estimated by determining the phosphorus content (13).

The homologs

intra-aortic elicited

bolus

RESULTS injection of

a dose-dependent

decrease

1342

the in

various left

AGEPC

ventricular

alkyl

chain

contractile

Vol.

161,

No.

3, 1989

BIOCHEMICAL

I5

b

I4

AND

I3

I2 -log

Pig. 1. Dose-response curves AGEPC and various AGEPC alkyl

BIOPHYSICAL

RESEARCH

II

[ AGON

IO

IST]

for the negative chain homologs

COMMUNICATIONS

9

8

moles

inotropic ((X6:0-.

effect C18:1-,

of beef-heart c15:0-, CU:O-

and C14:0-) in isolated guinea pig hearts. Ordinate: maximum changes in left ventricular contractile force from control. Points are means (+ S.E.; n = 57). Only one dose of AGEPC or AGEPC homologs was administered to each heart.

force

(Fig.

prompt

and

1)

and

reached

left

ventricular

80%.

In

contrast

concentrations

of

force

to

and coronary chain

C16:0-AGEPC

flow

the

for

are most

coronary

acetylated

alkyl

caused the

shown

only

of

flow chain

minimal

negative in

active

curves various maximum - 5-7).

are means (+ S.E.; n administered to each heart.

Fig.

these

responses

were

Maximum decreases in ranged between 60 and AGEPC

homologs,

decreases

inotropic 1 and

homolog,

-log

Fig. 2. Dose-response beef-heart AGEPC and pig hearts. Ordinate:

and

Both

2 minutes.

in

high

contractile

(3 and 15%, respectively).

curves

homologs was

the

2).

within

force

lyso-GEPC flow

(Fig.

levels

contractile

Dose-response alkyl

coronary maximal

[

AGONIST]

while

effect

the the

of various

EC50 values C14:0-AGEPC

in

AGEPC Table

and

C18:0-

moles

for the coronary-vasoconstricting effect of AGEPC alkyl chain homologs in isolated guinea changes in coronary flow from control. Points Only one dose of AGEPC or AGEPC homologs was

1343

1.

Vol.

161,

No.

BIOCHEMICAL

3, 1989

Table 1. EC50 values coronary-vasoconstricting

AND

BIOPHYSICAL

RESEARCH

for the myocardial-depressant and effects of alkyl chain homologs of PAF LVCFa

CFRb

C18:l AGEPC c15:o C18:O

1.5x10-13 2.0x10-12 1.2x10-11 3.5x10-11 1.0x10-10

c14:o

2.5x10-10

4 5x10-12 2 5x10-12 6 OxlO‘I2 6 ;;;;:;; 2 3 5x10-10

Comoound C16:O

All values are molar concentrations and are derived data reported in Figures 1 and 2. aLeft ventricular contractile force. bCoronary flow rate. AGEPC homologs

were

the

least

active.

units

Of interest,

beef-heart-derived

AGEPC, which

was

units

the

C16:0-AGEPC,

greater

The second

AGEPC, was 2 log of

COMMUNICATIONS

in potency

nearly

2 log

than

most either

less

from the

active

homolog,

C18:0-

or Cl5:0-AGEPC.

contained

C18:1-

approximately

active

than

in coronary

flow

70%

synthetic

C16:0-

AGEPC. Dose-response alkyl

chain

1.

the

AGEPC homologs

case

for

were

the

However,

in

beef-heart-derived C16:0-

for

AGEPC homologs

As was

AGEPC.

curves

the

are the

shown

negative

least

in Fig.

2 and the

inotropic

effect,

active

striking

molecular

contrast

AGEPC had

and C18:1-AGEPC

decrease

alkyl

to

biological chain

its

by various

EC50 values the

species,

C14:0-

by

inotropic

comparable

in Table and C18:0-

followed

negative

activity

caused

C15:0-

effect, to

that

here

had

the of

the

homologs. DISCUSSION

Our findings all

of

the

negative

inotropic

guinea the

pig

rank

C18:0-

order

For

= C18:1-

Several chain

increase the

chain. that

of

the

respect

fully

merit of

as seen

in

alkyl

chain

left

to

in

ventricular

> C18:1-

> C18:0-

to the rank

special

comment.

both

Figures

homolog

saturated

unsaturated 1 and was more

C18:0-AGEPC was

that

2,

homolog. the

1344

force, the

>

order

= C14:0-AGEPC.

order The

the

isolated

AGEPC > C15:0-

of potency first

double than

potent

contractile

> beef-heart

and coronary-vasoconstricting a single

the

coronary-vasoconstriction,

related

inotropic

observation,

in

AGEPC (4),

tested effects

AGEPC > C15:0-

features

AGEPC homologs

Indeed,

to beef-heart-derived

AGEPC homologs

decrease

= beef-heart

insertion

addition

was C16:0-

with

in negative

AGEPC unsaturated unexplained

the

interesting

simple

in chain

coronary-vasoconstricting

of potency

> C14:0-AGEPC;

alkyl

that, alkyl

and

heart.

was C16:0-

by

indicate

synthetic

is the marked potency obtained

bond

potency

2 log

units

A second,

beef-heart-derived

of the

in of

the the

alkyl C18:1-

greater and

than as yet

AGEPC

was

Vol.

161,

No.

3, 1989

significantly than

BIOCHEMICAL

less

potent

C16:0-AGEPC

beef-heart however,

the

comparable the

the

not

mechanisms of

is

Ca2+

current In

(Robertson,

decrease the

Furthermore,

our (4),

decrease

Pinckard in

previous

It

it

vasoconstriction

(2). that

be

argued

coronary-vasoconstricting differences

in

homologs

are

highly

cell-derived

plasma decrease the

be

in

recirculating play

minor

present

study

stimulation

and/or to the

Cl6:0-AGEPC ClS:O-

very a

source

to

bolus

system,

role

in

of

the

with

our

target cell stimulation

homologs

published

have

(PMN)

Indeed, alkyl

chain

of each

(5),

some of

the

AGEPC homologs

PAF receptor heterogeneity. of washed rabbit platelets, 0.14 identical ED5D's (i.e.,

homologs

are

only

1345

2-,

3-

PAF

either

plasma

current

model,

both drug

since

measured in

in

a non-

uptake

between

relationship

and neutrophils

these

PAF homologs that

SAR

regional

Furthermore, are

of

potencies

previously

the

enzyme.

differences

structure-activity

of these

and Cl4:0-AGEPC

In

anticipate

the

from and

Although

vitro.

injections

we would

determining

the

resulting

acetylhydrolase,

the

of

may reflect

uptake.

in

of

independent

myocardial-depressant

PAF homologs

enzyme

stable

and

component

ischemia

and coronary-vasoconstriction

response

differences.

of potency

respect

as

of platelets

interesting

species

the

cells.

inotropic

separate

in or

to the

are

excluded in

of

and coronary-vasoconstricting

Comparison

order

they

perfusion

a

depressant

and

susceptible

contractility

same heart

differences

presumably muscle

negative

a minor

local

the

vasoconstriction

the

PAF are

case,

intracellular

smooth

that

slow

by PAF in

observations),

that

biotransformation

(5),

can

the

in

arteriolar

indicated

the

either

elicited

Ca 2+ in vascular

potencies

cardiac

flow

unpublished

on

inotropic in

in

is

different

negative

reduction

with

depend

that

(6);

for

homologs

a decrease

cytosolic

may

could

the

to

chain

The

coronary

conceivable

potency

suggests

and Levi,

is

of

and

activity

associated

have

order

coronary-vasoconstricting

with

of in

responses

although

rank

responses. Na+

studies

the

activity

the

be associated

because

contractility

in

coronary

or

cardiac to

coronary-vasoconstricting

phenomena

agent

70% of

Paradoxically,

AGEPC alkyl for

surprising

is

an increase

the

various order

decrease

heart

McManus,

C16:0-AGEPC.

that

intracellular

would

from

since

2).

the the

two

and

pig

inotropic

unexpected

fact

apparent of

found

contrast,

guinea

resulting and

these

(14)

contractility calcium.

is

entirely

been

COMMUNICATIONS

as a negative was

in

(Fig.

to

not

PAF has

isolated

it effect

mediate

effect

is

RESEARCH

AGEPC had coronary-vasoconstricting

identical,

This

units)

finding

of C16:0-AGEPC

inotropic

but

action.

This

we utilized

preceding,

negative

similar,

BIOPHYSICAL

2 log

1).

beef-heart-derived

to that

From

(i.e.,

(Fig.

AGEPC which

AND

will

myocardial-

PAF homolog. (SAR)

data reveals

in

the

PAF-induced

several

differences suggest

data

(for

striking

in

the

the presence

rank of

For example, with both the Cl5:0and r&i), while the C18:1-,

and g-fold

less

active

(5).

Vol.

161,

No.

BIOCHEMICAL

3, 1989

However,

with

respect

log

more

potent

unit

than

3 log

C16:0-

units

and

effects;

to than

more

in

comparable human

the

increases

compared

with and

potency C18:0-AGEPC

C15:0-AGEPC

effects

less

(Figs. In

of

a rank

order

order

for

their

cardiac

of and

correlate

active

the

were

the

than

the

to

negative

more

On the have

their

AGEPC-induced and

unsaturated

other

in

units

inotropic

than

nearly

C16:0-AGEPC

2 log

despite

of a single by

(5).

1

and more

contrast,

1 to

respect

2 log hand,

the

PMN (5 and unpublished

present

AGEPC elicit

with

exception

with

homologs

C15:0In

resistance,

insertion

chain

than

was

double units

as

whereas

the

same potency

as

observations), terms

of

they

their

cardiac

1 and 2).

conclusion,

homologs

analogs

C18:1-AGEPC

human

C16:0-AGEPC

coronary-vasoconstricting

With

homolog

alkyl

stimulating

significantly

chain

keeping

of

COMMUNICATIONS

and C14:0-AGEPC.

vascular

SAR data,

the in

in

action,

more potent

activities. and

the

ClG:O-AGEPC are

alkyl

platelet-stimulating

RESEARCH

identical

coronary

coronary-vasoconstricting

C14:0-

had

other

increasing

units

ClG:O-

homologs

PMN stimulation,

bond

than

BIOPHYSICAL

inotropic

2 log

18:1-,

Cl8:1-AGEPC

active

negative

potent

nevertheless,

less

their

AND

of potency

that

in

is

left

with Thus,

and

effects their

similar,

these

by various

alkyl

chain

activation

could

be governed

of data

the

although the

alkyl

potency suggest

AGEPC homologs

various not

that

initiating

by unique

receptor

AGEPC homologs rabbit

occupation signal

force to the with

potency

stimulating

the

chain

identical, Notably,

relative

chain in

alkyl

contractile

effects.

ClS:l-AGEPC,

relative

that

ventricular

coronary-vasoconstricting

coronary

and human PMN.

demonstrates

a decrease

Cl6:0-AGEPC

well

study

the

for

the

did

not

platelets

of PAF receptors

transduction

and cell

specificities.

ACKNOWLEDGMENTS 37407,

This work was supported by U.S.Public HL 18828, HL 22555 and AI 21818.

Health

Service

Grants

HL 34215,

HL

REFERENCES 1. 2.

3. 4. 5. 6.

Alloatti, G., Montrucchio, G., Mariano, F., Tetta, C., DePaulis, R., Morea, M., Emanuelli, G. and Camussi, G. (1986) Int. Archs. Allergy appl. Immun. 79, 108-112. Levi, R., Shen, T.Y., Yee, S.J., Robertson, D.A., Genovese, A., Isom, O.W. and Krieger, K.H. (1987) In: New Horizons in Platelet Activating Factor Research, ed. by. C.M. Winslow and M.L. Lee, John Wiley & Sons, Ltd., Chichester, UK, 255-267. Robertson, D.A., Wang, D.-Y., Lee, C.O., Levi, R. (1987) J. Pharmacol. Exp. Ther. 245, 124-128. Levi, R., Burke, J.A., Guo, Z-G., Hattori, Y., Hoppens, C.M., McManus, D.J. and Pinckard, R.N. (1984) Circ. Res. 54, 117-124. L.M., Hanahan, Pinckard, R.N., Ludwig, J.C. and McManus, L.M. (1988) In: Inflammation Basic Principles and Clinical Correlates, ed. by J.I. Gallin, I.M. Goldstein and R. Snyderman, Raven Press, Ltd., New York, 139-166. Robertson, D.A., Genovese, A. and Levi, R. (1988) J. Pharmacol. Exp. Ther. 243, 834-839. 1346

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BIOCHEMICAL

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