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Catecholamines affect the binding of opioids to δ and K receptors in rat heart sarcolemmal membranes
J Mol
Cell
Cardiol21
(Supplement
II) (1989)
538
STIMULATORY EFFECT OF CATALYTIC SUBUNIT OF CYCLIC AMP-DEPENDENT PROTEIN KINASE ON LIPID METHYLATION IN ISOLATED CARDIAC SARCOLEMMA. R. Vetter, :‘. Panagia, J. Dai, Y. Taira, N.S. Dhalla. Division of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, and Departments of Physiology and Anatomy, Faculty of Medicine, University of Manitoba, Winnipeg, Canada. Purified sarcolemmal (SL) membranes, from both rat and pig myocardium exhibit increased methyltransferase activity after preincubation with MgATP and catalytic subunit of cyclic AMP-dependent protein kinase. Prephosphorylation of membranes stimulates c3Hl-methyl incorporation into SL lipids assayed at 0.15 uM S-adenosyl-L-[methyl-3H1 methionine ([3H]-AdoMet) 2.4- and 1.4- fold in rat and pig heart membrane preparations, respectively. Kinetic analysis of the methylation reaction reveals that the stimulation is due to an enhancement of Vmax. The Km of the high-affinity methylation reaction, ranging from 0.08 to 0.11 uM, is not changed under phosphorylating conditions. Premethylation of SL in the presence of 0.15 to 150 uM AdoMet did not change the phosphorylation of SL substrates catalyzed either by endogenous cyclic AMP-dependent protein kinase or by added catalytic This study suggests that cardiac SL methyltransferase@, which is (are) involved subunit. in the covalent modification of membrane lipids, can be controlled by cyclic AMP-dependent protein phosphorylation. It indicates also that the lipid composition of the sarcolemma may be under beta-adrenergic control. (Supported by a grant from the Medical Research Council of Canada).
5%
EFFECTS A. Pentz, Stellenbosch of South
OF METABOLIC I.S. Harper. Medical Africa.
MANIPULATION MRC Centre School; MRC
ON MYOCARDIAL for Molecular Institute for
SARCOLEMMAL PERMEABILITY. A. and Cellular Biology, University Medical Biophysics, Tygerberg,
Lochner, of Republic
The harmful effects of free fatty acids and the protection afforded by glucose on the ischaemic heart are well documented. However, the mechanisms involved in these pro= cesses are poorly understood. To determine whether metabolic manipulation of the sub= thereby impairing intracel= strate supply act through altered sarcolemmal permeability, lular ionic homeostasis, isolated rat hearts were subjected to control and hypoxic low-flow perfusion using glucose, palmitate or glucose plus palmitate as substrates. Sarcolemmal permeability was quantified using an ultrastructural probe (ionic lantha= num) as well as LDH release. Palmitate (0.5 mM, palmitate/albumin molar ratio 5) as well as substrate-free perfusion increased lanthanum influx, LDH release and intracel= lular Ca2+ levels in hypoxic, low-flow perfused hearts. Glucose (10 mM) as substrate afforded significant protection against ischaemic damage as evidenced by a reduction of al I three above-mentioned parameters. In summary, palmitate as substrate can cause . . ;;.e;re sarcolemmal Injury, whereas glucose reduces membrane impairment and cellular overloadrng. These substrate effects were associated with marked changes in sar= colemmal phospholipid-fatty acid composition, which could be the cause of the increased membrane permeab i I i ty .
540
CATECHOLAMINES
AFFECT
THE
BINDING
C.
Ventura,
L.
Bastagli,
P.
of
Research
on
Cardiac
Metabolism,
Thj using
apioid
[
gene
284.3
and,
to
the
prot the
agonist
value
This
effect
in
KD
value.
phentolamine
ly
inhibited
+
stimulated
was
associated
(1
by
DADLE of
opioid
catecholamine
and,
sarcolemmal
a radioligand.
nM
prot)
displaced
with
produced
to
a
receptors
interaction
lower
6
of with
a
Emax
and
extent,
by
and
K
and
R
MEMBRANES.
Biochemistry,
Centre
2
paralleled
the
KD
and
R-agonists
30
type sarcolemmal
and
Bmax as
by
was
the
vajue [
results
indicate
that
adrenoceptors.
and
values DADLE
HI-DPN
phenyl-
an
increase
2
0.1
higher without
completely
opioidergic
whilst
a-agonist
(1.3
demonstrate this
K
agoni:t
6
prat) was
by of
manner,
opioid These
the
by
fmol/mg
R-adrenergic-stimulated U50.48BH.
S.180
a-
basal
of
(610.5
ventricles concentrations
dose-dependent
concentrations
value the
in
at
well
stimulated effect
decrease at
The
a
heart
found
as
in
H]-DPN
an
rat
was
DPN,
markedly
a
both
D-
[
Such
only
the
by The
respectively.
SARCOLEMMAL of
binding.
was
-DPN.
binding
in
increase
action
H]
HEART
from
binding
bound
with
the
an
isolated
cooperative
binding
[
RAT
Department
Unljbeled
together
increased
PM)
a
Thq
IN
Saturable
respectively.
nM
RECEPTORS
Guarnieri.
membranes
suggested 0.3
K C.
Italy.
in
0.5-20
fmol/ng
AND and
as
ineffective. of
6
Bologna,
lJ50,4BBH,
was
propranolol
of
+
agonist
stimulatory and
by
K
38
TO Caldarera
analysis 4.2
presence
(988.4
The
sarcolemma,
HI-DPN)
and
DAGO
the
isoproterenal
by
[
Scatchard
The
fmol/mg extent
vM)
Bmax
D-agonist
the
H]-DPN. 20
lower
(1
gerfarmed
(
p selective
the
the
+ a
ephrine
nM.
[
nM
OPIOIDS C.M.
University
was
H]-Digrenorphine
20
about
binding
OF
Bernardi,
in
nM).
The
than
2.5
altering antagonized
binding
was the
partial-
in
presence, system
nay
be
Cell
Cardiol21
(Supplement
II) (1989)
538
STIMULATORY EFFECT OF CATALYTIC SUBUNIT OF CYCLIC AMP-DEPENDENT PROTEIN KINASE ON LIPID METHYLATION IN ISOLATED CARDIAC SARCOLEMMA. R. Vetter, :‘. Panagia, J. Dai, Y. Taira, N.S. Dhalla. Division of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, and Departments of Physiology and Anatomy, Faculty of Medicine, University of Manitoba, Winnipeg, Canada. Purified sarcolemmal (SL) membranes, from both rat and pig myocardium exhibit increased methyltransferase activity after preincubation with MgATP and catalytic subunit of cyclic AMP-dependent protein kinase. Prephosphorylation of membranes stimulates c3Hl-methyl incorporation into SL lipids assayed at 0.15 uM S-adenosyl-L-[methyl-3H1 methionine ([3H]-AdoMet) 2.4- and 1.4- fold in rat and pig heart membrane preparations, respectively. Kinetic analysis of the methylation reaction reveals that the stimulation is due to an enhancement of Vmax. The Km of the high-affinity methylation reaction, ranging from 0.08 to 0.11 uM, is not changed under phosphorylating conditions. Premethylation of SL in the presence of 0.15 to 150 uM AdoMet did not change the phosphorylation of SL substrates catalyzed either by endogenous cyclic AMP-dependent protein kinase or by added catalytic This study suggests that cardiac SL methyltransferase@, which is (are) involved subunit. in the covalent modification of membrane lipids, can be controlled by cyclic AMP-dependent protein phosphorylation. It indicates also that the lipid composition of the sarcolemma may be under beta-adrenergic control. (Supported by a grant from the Medical Research Council of Canada).
5%
EFFECTS A. Pentz, Stellenbosch of South
OF METABOLIC I.S. Harper. Medical Africa.
MANIPULATION MRC Centre School; MRC
ON MYOCARDIAL for Molecular Institute for
SARCOLEMMAL PERMEABILITY. A. and Cellular Biology, University Medical Biophysics, Tygerberg,
Lochner, of Republic
The harmful effects of free fatty acids and the protection afforded by glucose on the ischaemic heart are well documented. However, the mechanisms involved in these pro= cesses are poorly understood. To determine whether metabolic manipulation of the sub= thereby impairing intracel= strate supply act through altered sarcolemmal permeability, lular ionic homeostasis, isolated rat hearts were subjected to control and hypoxic low-flow perfusion using glucose, palmitate or glucose plus palmitate as substrates. Sarcolemmal permeability was quantified using an ultrastructural probe (ionic lantha= num) as well as LDH release. Palmitate (0.5 mM, palmitate/albumin molar ratio 5) as well as substrate-free perfusion increased lanthanum influx, LDH release and intracel= lular Ca2+ levels in hypoxic, low-flow perfused hearts. Glucose (10 mM) as substrate afforded significant protection against ischaemic damage as evidenced by a reduction of al I three above-mentioned parameters. In summary, palmitate as substrate can cause . . ;;.e;re sarcolemmal Injury, whereas glucose reduces membrane impairment and cellular overloadrng. These substrate effects were associated with marked changes in sar= colemmal phospholipid-fatty acid composition, which could be the cause of the increased membrane permeab i I i ty .
540
CATECHOLAMINES
AFFECT
THE
BINDING
C.
Ventura,
L.
Bastagli,
P.
of
Research
on
Cardiac
Metabolism,
Thj using
apioid
[
gene
284.3
and,
to
the
prot the
agonist
value
This
effect
in
KD
value.
phentolamine
ly
inhibited
+
stimulated
was
associated
(1
by
DADLE of
opioid
catecholamine
and,
sarcolemmal
a radioligand.
nM
prot)
displaced
with
produced
to
a
receptors
interaction
lower
6
of with
a
Emax
and
extent,
by
and
K
and
R
MEMBRANES.
Biochemistry,
Centre
2
paralleled
the
KD
and
R-agonists
30
type sarcolemmal
and
Bmax as
by
was
the
vajue [
results
indicate
that
adrenoceptors.
and
values DADLE
HI-DPN
phenyl-
an
increase
2
0.1
higher without
completely
opioidergic
whilst
a-agonist
(1.3
demonstrate this
K
agoni:t
6
prat) was
by of
manner,
opioid These
the
by
fmol/mg
R-adrenergic-stimulated U50.48BH.
S.180
a-
basal
of
(610.5
ventricles concentrations
dose-dependent
concentrations
value the
in
at
well
stimulated effect
decrease at
The
a
heart
found
as
in
H]-DPN
an
rat
was
DPN,
markedly
a
both
D-
[
Such
only
the
by The
respectively.
SARCOLEMMAL of
binding.
was
-DPN.
binding
in
increase
action
H]
HEART
from
binding
bound
with
the
an
isolated
cooperative
binding
[
RAT
Department
Unljbeled
together
increased
PM)
a
Thq
IN
Saturable
respectively.
nM
RECEPTORS
Guarnieri.
membranes
suggested 0.3
K C.
Italy.
in
0.5-20
fmol/ng
AND and
as
ineffective. of
6
Bologna,
lJ50,4BBH,
was
propranolol
of
+
agonist
stimulatory and
by
K
38
TO Caldarera
analysis 4.2
presence
(988.4
The
sarcolemma,
HI-DPN)
and
DAGO
the
isoproterenal
by
[
Scatchard
The
fmol/mg extent
vM)
Bmax
D-agonist
the
H]-DPN. 20
lower
(1
gerfarmed
(
p selective
the
the
+ a
ephrine
nM.
[
nM
OPIOIDS C.M.
University
was
H]-Digrenorphine
20
about
binding
OF
Bernardi,
in
nM).
The
than
2.5
altering antagonized
binding
was the
partial-
in
presence, system
nay
be