(Circulation. 1995;91:2226-2234.)
© 1995 American Heart Association, Inc.
Articles |
1-Adrenoceptor Activation Increases Ecto-5'-Nucleotidase Activity and Adenosine Release in Rat Cardiomyocytes by Activating Protein Kinase C
From the First Department of Medicine, Osaka University School of Medicine, Suita; Cardiology, Department of Internal Medicine, Sakurabashi Watanabe Hospital, Osaka (K.I.); and the Department of Medical Information Science, Osaka University Hospital, Suita (M.I.), Japan.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background Adenosine is an important regulator of many cardiac functions and is synthesized primarily by ecto- and cytosolic 5'-nucleotidase. We have previously reported that
1-adrenoceptor blockade attenuates adenosine release
from ischemic myocardium, raising the possibility that
1-adrenoceptor activation activates 5'-nucleotidase.
This study tested whether activation of protein kinase C by
1-adrenoceptor activation increases 5'-nucleotidase
activity and augments adenosine release.
Methods and Results Cardiomyocytes were isolated from adult male
Wistar rats and suspended in modified HEPES-Tyrode's buffer solution.
After stabilization, the cardiomyocytes were incubated with and without
an exposure to norepinephrine (10-9 to 10-5
mol/L) while being treated with propranolol and yohimbine or with and
without an exposure to methoxamine (10-9 to
10-5 mol/L). Ecto-5'-nucleotidase activity was
increased
by norepinephrine and methoxamine during 30 minutes in a dose-dependent
manner, whereas cytosolic 5'-nucleotidase was not activated. These
increases in ecto-5'-nucleotidase activity were inhibited by
GF109203X, an inhibitor of protein kinase C, and mimicked by phorbol
12-myristate 13-acetate (PMA), an activator of protein kinase C. The
increase in ecto-5'-nucleotidase was not prevented by cycloheximide.
When ecto-5'-nucleotidase activity increased, adenosine release was
augmented in methoxamine- and PMA-treated cardiomyocytes (1299±252%
and 1372±149%, respectively) compared with the untreated group
(578±26%). The increase in adenosine release was blunted by GF109203X
and ,ß-methyleneadenosine 5'-diphosphate, an inhibitor of
ecto-5'-nucleotidase.
Conclusions Thus, we conclude that
1-adrenoceptor–mediated increases in
ecto-5'-nucleotidase activity are attributed to activation of protein
kinase C in rat cardiomyocytes.
Key Words: nucleotides • norepinephrine • ischemia • hypoxia
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Adenosine has been reported to modify several key cellular activities in the heart.1 2 3 Adenosine relaxes vascular smooth muscles,4 inhibits platelet aggregation5 6 and oxygen-derived free radical generation from polymorphonuclear leukocytes,7 and attenuates increases in myocardial contractility due to norepinephrine.8 9 In the heart, adenosine is released from cardiomyocytes, coronary endothelial and coronary smooth muscle cells, and leukocytes.2 3 During ischemia and hypoxia, adenosine is released from cardiomyocytes primarily through 5'-nucleotidase. This enzyme is known to be modulated by myocardial cellular metabolic factors, eg, ATP, ADP, and inorganic phosphate (Pi)10 11 12 : Ecto-5'-nucleotidase is inhibited by ATP and ADP,10 whereas cytosolic 5'-nucleotidase is activated by ATP and ADP and inhibited by Pi.11 12 On the other hand, norepinephrine is released from presynaptic vesicles in the ischemic heart,13 which may activate
1-adrenoceptors
and protein kinase C through accumulation of diacylglycerol.
Furthermore, myocardial ischemia translocates protein kinase C from the
cytosol to the plasma membrane.14 These two activities may
synergistically activate protein kinase C in the cardiomyocytes during
ischemia. We previously reported that 1-adrenoceptor
activity regulates release of adenosine,15 16 raising
the
possibility that 1-adrenoceptor activation may increase
5'-nucleotidase activity by activating protein kinase
C.17
To test this hypothesis, we measured both ecto- and cytosolic
5'-nucleotidase activity and adenosine release with and without
exposure to norepinephrine, methoxamine, and phorbol 12-myristate
13-acetate (PMA). We also tested whether the increase in
5'-nucleotidase activity is blocked by GF109203X and
,ß-methyleneadenosine 5'-diphosphate (AOPCP). In addition, we
examined whether augmentation of 5'-nucleotidase activity increases
adenosine release from rat cardiomyocytes.
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Methods |
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Materials
Adenosine, norepinephrine, propranolol, yohimbine, PMA, AOPCP, and cycloheximide were obtained from Sigma Biochemical Co. GF109203X was obtained from Calbiochem. 2'-Deoxycoformycin was provided by Yamasa Shoyu Research Laboratories. 5'-Iodotubercidin was obtained from Research Biochemicals Inc. All reagents and chemicals were the highest grade obtainable.
Preparation of Cardiomyocytes
Cardiomyocytes were isolated
from 128 adult male Wistar rats
(200 to 250 g) as described previously.18 Briefly, heparin
(600 U IP) was injected 30 minutes before anesthesia with pentobarbital
(0.2 mg/g body wt IP). The hearts were removed quickly and perfused in
a Langendorff perfusion apparatus, initially with 25 mL of
Ca2+-free modified HEPES-KOH/Tyrode's buffer
solution (in mmol/L: NaCl 120, KCl 5, MgSO4 1.2,
NaHCO3 5, glucose 10, and HEPES 20; pH 7.3) equilibrated
with 95% O2/5% CO2 at 37°C and then
with digestion solution (Ca-free solution containing 0.06 wt% crude
collagenase and 0.1% fatty acid–free bovine albumin) for 30 minutes.
The ventricle was removed and finely minced with scissors in
Ca2+-containing HEPES/Tyrode's solution (1 mmol/L
CaCl2). The cardiomyocytes were mechanically dispersed by
gentle pipetting, filtered through gauze, and suspended in
Ca2+-containing solution. Cells were washed twice
with Ca2+-containing solution, collected by
centrifugation (50g, 1 minute), and resuspended in
Ca2+-containing solution. Microscopy demonstrated
that almost all of the isolated cells were cardiomyocytes (2% of
the cellular components resembled fibroblasts) and that 86±4% of the
isolated cardiomyocytes were morphologically (rod shaped) and
metabolically (trypan blue exclusion test) intact.
Experimental Protocols
Protocol 1: Effects of
Norepinephrine, Methoxamine, and PMA on
Ecto- and Cytosolic 5'-Nucleotidase Activity
After
cardiomyocytes were isolated from rats (n=72),
10-6 mol/L norepinephrine was added to the suspension of
1.0 to 2.0x107 cells. Cardiomyocytes obtained from
different rats were used in each experiment. Propranolol
10-5 mol/L and yohimbine 10-5 mol/L were
added to the suspension 10 minutes before its exposure to
norepinephrine. In a preliminary study, we confirmed that neither
propranolol nor yohimbine affects ecto- and cytosolic 5'-nucleotidase
activity (data not shown). First, we recorded sequential changes in
ecto- and cytosolic 5'-nucleotidase activity after exposure to
10-6 mol/L norepinephrine (n=5). Next, we varied the
concentration of norepinephrine from 10-9 to
10-5 mol/L and measured ecto- and cytosolic
5'-nucleotidase activity at 15 minutes after exposure to norepinephrine
(n=5 in each dose). This 15-minute interval was necessary to reach a
steady state for ecto- and cytosolic 5'-nucleotidase activity (Fig
1
). To test whether activation of ecto-5'-nucleotidase
is attributable to activation of protein kinase C, the effect of
GF109203X (10-7 mol/L) on the dose-response curve between
norepinephrine and 5'-nucleotidase activity (n=5 in each dose of
norepinephrine) was observed. Furthermore, to test whether protein
synthesis is involved in activation of ecto-5'-nucleotidase, the effect
of cycloheximide (1.4x10-6 g/mL), a blocker of protein
synthesis, on the dose-response curves between norepinephrine and
5'-nucleotidase activity (n=5 in each dose of norepinephrine) was
observed.
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Furthermore, we tested the effect of an
1-adrenoceptor
agonist, methoxamine, and an activator of protein kinase C, PMA. First,
we recorded sequential changes in ecto- and cytosolic 5'-nucleotidase
activity after exposure to 10-6 mol/L methoxamine
(n=5)
and 10-7 mol/L PMA (n=5). Next, we varied the
concentrations of methoxamine from 10-9 to
10-5 mol/L and PMA from 10-10 to
10-6 mol/L and measured ecto- and cytosolic
5'-nucleotidase activity at 15 minutes after an exposure to methoxamine
and PMA (n=5 in each dose). This 15-minute interval was necessary to
reach a steady state for ecto- and cytosolic 5'-nucleotidase activity
(Figs 2-4). To test whether activation of
ecto-5'-nucleotidase is attributable to the activation of protein
kinase C, the effect of GF109203X (5x10-7 mol/L) on the
dose-response curves between methoxamine and PMA and
ecto-5'-nucleotidase activity (n=5 in each dose of methoxamine
and PMA)
was observed. GF109203X is a specific inhibitor of protein kinase C. To
test whether protein synthesis is involved in increases in
ecto-5'-nucleotidase activity, the effect of cycloheximide
(1.4x10-6 g/mL) on the dose-response curves between
methoxamine and PMA and ecto-5'-nucleotidase activity (n=5 in
each dose
of methoxamine and PMA) was observed.
To determine whether norepinephrine, methoxamine, and PMA activate protein kinase C of the membrane fraction, we examined the time courses of changes in protein kinase C activity in the membrane and cytosolic fractions in the groups of norepinephrine (10-6 mol/L, n=5), methoxamine (10-6 mol/L, n=5), and PMA (10-7 mol/L, n=5) treatments. Since we found that activation of protein kinase C reaches a steady state in 15 minutes, this 15-minute interval was necessary to reach a steady state for protein kinase C activity. Therefore, we examined the dose-response relation between norepinephrine (10-9 to 10-5 mol/L, n=5 in each dose), methoxamine (10-9 to 10-5 mol/L, n=5 in each dose), and PMA (10-10 to 10-6 mol/L, n=5 in each dose) and protein kinase C activity at 15 minutes after exposure to each chemical.
Protocol 2: Release of Adenosine and 5'-AMP
From Rat Cardiomyocytes
Pretreated With and Without Norepinephrine, PMA, and GF109203X
In this
protocol, cardiomyocytes were isolated from 56 adult
male Wistar rats. Cardiomyocytes obtained from the different rats were
used in each experiment. To verify that increases in
ecto-5'-nucleotidase activity are involved in augmented adenosine
release under normoxic and hypoxic conditions, we measured adenosine in
the medium of the suspension with 1.0 to 2.0x107 rat
cardiomyocytes pretreated with 10-6 mol/L of methoxamine
or 10-7 mol/L of PMA with and without exposure to
10-5 mol/L of GF109203X for 30 minutes. Furthermore, to
examine whether 5'-AMP, a substrate for ecto-5'-nucleotidase to
produce
adenosine, is also released from cardiomyocytes, we measured 5'-AMP in
the medium of the suspension of cardiomyocytes at 15 minutes in each
treatment. The cells were gently washed twice and were incubated in
normoxic or hypoxic solutions bubbled with 95%
O2/5% CO2 and 95%
N2/5% CO2, respectively. The
PO2 was 478±8 and 32±6 mm Hg,
respectively, in the two solutions. To test the cause-and-effect
relation between the ecto-5'-nucleotidase activation and the augmented
adenosine release, we examined the effect of AOPCP (50 µmol/L) in all
the groups. AOPCP was added after the baseline collection of suspension
for adenosine. We made the cardiomyocytes hypoxic after the
baseline measurements and during addition of AOPCP. The groups
consisted of (1) no treatment, (2) methoxamine pretreatment, (3) PMA
pretreatment, (4) methoxamine and GF109203X pretreatment, (5) PMA and
GF109203X pretreatment, (6) AOPCP treatment, (7) methoxamine
pretreatment with AOPCP treatment, and (8) PMA pretreatment with AOPCP
treatment groups (n=5 in each group). Sequential changes in adenosine
concentration were recorded after exposures to 10 µmol/L
2'-deoxycoformycin to inhibit adenosine deaminase and 1 µmol/L
5'-iodotubercidin to inhibit adenosine kinase.
Adenosine and 5'-AMP Measurements
Details of the
methods used to measure adenosine and 5'-AMP
concentrations have been
reported.15 16 19 20 To
measure
sequential changes in adenosine release from cardiomyocytes, we
obtained the medium of the suspension of cardiomyocytes by
centrifugation at 50g for 1 minute. We added 500 mmol/L EDTA
and 10% trichloroacetic acid to the medium to inhibit degradation of
adenosine and 5'-AMP. Trichloroacetic acid was removed by
water-saturated ether. After centrifugation (1000g), the
supernatant was collected and the adenosine and 5'-AMP concentrations
were determined.
Adenosine in the solution (100 µL) was treated with succinic acid anhydride and trimethylamine in 100 µL of dioxane. After a 20-minute incubation at 37°C, the mixture was diluted with 100 µL of adenosine 2',3'-O-disuccinyl-3-[125I]iodotyrosine methyl ester (0.5 pmol) and 100 µL of diluted antiadenosine serum. The mixture was kept in a cold-water (4°C) bath for 18 hours, and a second antibody solution (500 µL goat anti-rabbit immunoglobulin G antiserum) was added. After incubation for 1 hour at 4°C and centrifugation for 20 minutes at 2500g, the supernatant was removed and radioactivity remaining in the tube was countered in a gamma counter. We stored the samples obtained from the medium in which the cardiomyocytes were suspended.
The concentration of 5'-AMP was also determined by a UV method described previously.20 We used the same medium of cardiomyocytes for measurement of adenosine.
We stored the samples at -80°C until use for each assay.
Measurements of Activities of 5'-Nucleotidase and Protein
Kinase
C
On completion of the experiments, the rat cardiomyocytes were
homogenized with a Potter-Elvehjem homogenizer (30 strokes) at 4°C
for 5 minutes in 10 volumes of ice-cold 10 mmol/L HEPES-KOH buffer (pH
7.4) containing 0.25 mol/L sucrose, 1 mmol/L MgCl2,
and 1 mmol/L mercaptoethanol. The crude homogenate was strained through
a double-layer nylon sieve and homogenized again for 1 minute. For
preparation of the crude membrane fraction, part of the homogenate was
centrifuged at 1000g at 4°C for 10 minutes. The resultant
pellet was washed three times and finally resuspended in the HEPES-KOH
buffer. To prepare the cytosolic fraction, the remainder of the
homogenate was centrifuged at 3000g at 4°C for 10 minutes,
and the supernatant was centrifuged at 200 000g at 4°C
for 1 hour. The membrane and cytosolic fraction were dialyzed at 4°C
for 4 hours against HEPES-KOH containing 1 mmol/L
MgCl2, 1 mmol/L mercaptoethanol, and 0.01%
activated charcoal and divided into aliquots that were frozen
immediately and stored at -80°C.
The activity of 5'-nucleotidase was measured by an enzymatic assay technique21 and is reported in units of nmol · mg protein-1 · min-1. 5'-Nucleotidase activity of membrane and cytosolic fractions were defined as ecto- and cytosolic 5'-nucleotidase activity, respectively. When cytosolic 5'-nucleotidase activity was measured, AOPCP (50 µmol/L) was added to inhibit contaminating ecto-5'-nucleotidase.
The activity of protein kinase C was measured by the enzyme assay using the RPN 77A kit (Amersham), which provides a simple and reliable method of estimating protein kinase C without extensive purification of the samples. Activity of protein kinase C was expressed as nmol · mg protein-1 · min-1. Protein concentrations were measured by the methods of Lowry et al22 using bovine serum albumin as a standard. Furthermore, to examine the Ca2+ and phospholipid dependencies of activity of protein kinase C, we measured protein kinase C activity adding 0.5 mmol/L in excess of EGTA to chylate Ca2+ and eliminating phosphatidyl-L-serine from the assay system.
Statistical Analysis
Statistical analysis was performed with
paired and unpaired
t tests.23 Repeated-measures ANOVA followed by
modified Bonferroni's multiple comparison were also performed to
evaluate intergroup differences in time courses and dose-response
curves. All values were expressed as mean±SEM, and P<.05
was considered significant.
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Results |
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Norepinephrine 10-6 mol/L increased ecto-5'-nucleotidase activity by 54.8±8.8% from 6.18±0.42 nmol · mg protein-1 · min-1 at 30 minutes (Fig 1
, top). Cytosolic 5'-nucleotidase activity
(control
value, 1.13±0.10 nmol · mg
protein-1 · min-1) was not increased
by
exposure to 10-6 mol/L norepinephrine (Fig 1,
bottom).
Methoxamine 10-6 mol/L also increased
ecto-5'-nucleotidase
activity by 53.0±10.3% from 5.95±0.85 nmol · mg
protein-1 · min-1 at 30 minutes (Fig
2,
top); however, cytosolic 5'-nucleotidase activity (control value,
1.01±0.12 nmol · mg
protein-1 · min-1) was not increased
by
exposure to 10-6 mol/L methoxamine (Fig 2,
bottom). The
time courses of increases in ecto-5'-nucleotidase activity were similar
between the norepinephrine- and methoxamine-treated groups,
and increases in ecto-5'-nucleotidase activity reached a steady state
at 15 minutes after exposure to each chemical. Ecto-5'-nucleotidase
activity showed a positive dose-response relation to norepinephrine and
methoxamine at concentrations between 10-9 and
10-5 mol/L (Fig 5). GF109203X inhibited the
dose-dependent increases in ecto-5'-nucleotidase activity due to
norepinephrine and methoxamine (10-9 to 10-5
mol/L in each) (Fig 5). However, cycloheximide did not inhibit
the
increases in ecto-5'-nucleotidase due to norepinephrine and methoxamine
(Fig 5). On the other hand, exposure to 10-7
mol/L
PMA increased ecto-5'-nucleotidase activity by 56.2±9.2% from
6.63±1.69 nmol · mg
protein-1 · min-1 at 30 minutes (Fig
3,
top); however, cytosolic 5'-nucleotidase activity (control value,
0.97±0.23 nmol · mg
protein-1 · min-1) was not increased
(Fig 3, bottom). A positive dose-dependent relation between PMA
and
ecto-5'-nucleotidase was demonstrated (Fig 4). This
activation of
ecto-5'-nucleotidase activity was inhibited by GF109203X and was not
affected by cycloheximide. These results indicate that (1)
1-adrenoceptor activation increases activity of
ecto-5'-nucleotidase, (2) the
1-adrenoceptor–mediated
increases in ecto-5'-nucleotidase activity are attributable to
activation of protein kinase C, and (3) activation of
ecto-5'-nucleotidase is independent of protein synthesis.
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To investigate whether norepinephrine, methoxamine, and PMA
increase the protein kinase C activity of the cardiomyocytes, we
measured protein kinase C activity of the membrane and cytosolic
fraction exposures to norepinephrine, methoxamine, and PMA with and
without concomitant exposures to GF109203X. Fig 6 shows
the time courses of the increases in the protein kinase C activity of
the membrane and cytosolic fractions and the summation of both
fractions after exposures to norepinephrine, methoxamine, and PMA. The
protein kinase C activity in the membrane fraction was increased in 10
minutes after exposures to norepinephrine, methoxamine, and PMA, which
preceded the activation of ecto-5'- nucleotidase. The extents of
increases in protein kinase C activity 30 minutes after exposures to
norepinephrine, methoxamine, and PMA were 88±16%, 99±19%, and
54±15%, respectively. In contrast, the protein kinase C activity of
the cytosolic fraction did not increase after exposures to
norepinephrine, methoxamine, and PMA. Fig 7 shows
the dose-response relations between the doses of norepinephrine,
methoxamine, and PMA with and without concomitant exposures
to GF109203X and the protein kinase C activity of the membrane and
cytosolic fractions and summation of both fractions. Norepinephrine and
methoxamine 10-9 to 10-5 mol/L and PMA
10-10 to 10-6 mol/L increased protein kinase
C activity of the membrane fraction in a dose-dependent manner. Table
1 shows the Ca2+- and
phospholipid-dependent activation of protein kinase C. Depletion of
Ca2+ and/or phospholipid decreased the basal
activity of protein kinase C of the cytosolic fraction but not the
basal activity of protein kinase C of the membrane fraction. On the
other hand, depletion of Ca2+ and/or phospholipid
blunted the activation of protein kinase C of the membrane but not of
the cytosol fraction. Thus, the increase in protein kinase C of the
membrane fraction after exposure to methoxamine and PMA was dependent
on both Ca2+ and phospholipid.
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Although we showed that ecto-5'-nucleotidase in rat cardiomyocytes is
activated by 1-adrenoceptor activation through increased
activity of protein kinase C in the membrane fraction, it is not clear
whether the activation of ecto-5'-nucleotidase increases release of
adenosine from cardiomyocytes. Therefore, we measured the adenosine
concentration of suspension with isolated normoxic cardiomyocytes
pretreated with and without a 30-minute exposure to 10-6
mol/L methoxamine and 10-7 mol/L PMA, with and without
GF109203X or AOPCP. There were no differences in adenosine
concentration in each group before additions of 2'-deoxycoformycin and
5'-iodotubercidin (basal adenosine concentration in the suspension
[pmol/mg protein]: control group, 4.10±0.54;
methoxamine-pretreated
group, 4.55±0.52; PMA-pretreated group, 4.26±0.69; methoxamine
and
GF109203X group, 4.53±0.42; PMA and GF109203X group, 4.24±0.36;
AOPCP
group, 4.75±0.68; methoxamine and AOPCP group, 4.38±0.51; and
PMA and
AOPCP group, 4.49±0.49). Adenosine release was augmented in the
methoxamine- and PMA-pretreated groups compared with the controls in
the normoxic conditions (Fig 8). Furthermore, this
augmentation of adenosine release was blunted by concomitant exposures
to GF109203X in the control levels. AOPCP decreased the accumulation of
adenosine, indicating that ecto-5'-nucleotidase contributed to
adenosine release. Interestingly, concomitant exposures to AOPCP in the
methoxamine- and PMA-pretreated groups blunted the increases in
adenosine release to the levels of the AOPCP-alone group, indicating
that enhanced release of adenosine in the methoxamine- and
PMA-pretreated groups is attributable to the activation of
ecto-5'-nucleotidase. Additionally, adenosine release from hypoxic
cardiomyocytes was higher after pretreatment with methoxamine and PMA
than in controls (Fig 9). The augmentation of adenosine
release due to PMA was also blunted by concomitant exposures to
GF109203X. There were no differences in adenosine concentration in each
group before additions of 2'-deoxycoformycin and 5'-iodotubercidin
and
hypoxic procedure (basal adenosine concentration in the solution
[pmol/mg protein]: control group, 3.43±0.37;
methoxamine-pretreated
group, 4.62±0.81; PMA-pretreated group, 4.44±0.64; methoxamine
and
GF109203X group, 4.52±0.34; PMA and GF109203X group, 4.23±0.25;
AOPCP
group, 4.78±0.65; methoxamine and AOPCP group, 4.62±0.76; and
PMA and
AOPCP group, 4.42±0.53). Table 2 shows the 5'-AMP
concentrations of the medium in which the cardiomyocytes were
suspended. In the normoxic condition, we detected 5'-AMP in the medium,
and the hypoxic condition increased the accumulation of 5'-AMP
concentration in the medium. There were no significant differences in
5'-AMP concentration between all of the groups in normoxic and hypoxic
cardiomyocytes.
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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Effects of Activation of Protein Kinase C on Ecto-5'-Nucleotidase Activity and Adenosine Release
The present study demonstrates that activation of protein kinase C during
1-adrenoceptor stimulation increases
ecto-5'-nucleotidase activity in rat cardiomyocytes and thereby
contributes to increases in adenosine release from normoxic and hypoxic
cardiomyocytes. Since increases in ecto-5'-nucleotidase activity due to
the activation of protein kinase C do not necessarily result in
increased adenosine release from rat cardiomyocytes, we had to
consider the cause-and-effect relation between the increases in
ecto-5'-nucleotidase activity and adenosine release. The major pathways
of adenosine synthesis include enzymatic dephosphorylation of 5'-AMP by
5'-nucleotidase and the hydrolysis of
S-adenosylhomocysteine (SAH) by
SAH-hydrolase.24 5'-Nucleotidase exists in two forms in
the myocardium: membrane-bound (ecto-5'-nucleotidase) and free in the
cytoplasm (cytosolic 5'-nucleotidase), and both are capable of
producing adenosine.25 26 27 Several
lines of evidence have
demonstrated that both ecto- and cytosolic 5'-nucleotidase are
essential for the production of adenosine in the hypoxic
cardiomyocytes.12 27 28 The present study
reveals that
ecto-5'-nucleotidase is more important for the pathway to increase
adenosine release in rat cardiomyocytes, because (1)
ecto-5'-nucleotidase activity was increased without altering cytosolic
5'-nucleotidase activity when release of adenosine was increased and
(2) AOPCP, an inhibitor of ecto-5'-nucleotidase, blunted the increases
in adenosine release from the rat cardiomyocytes (Figs 8 and
9). The
present result is consonant with the work of Imai et
al29 showing that ecto-5'-nucleotidase plays an essential
role in releasing adenosine in hypoxic cardiomyocytes. In turn, Lloyd
and Schrader30 showed that adenosine production by
SAH-hydrolase is also essential in the normoxic condition, and we do
not disagree with this. During ischemia and hypoxia, the contribution
of SAH-hydrolase to adenosine release is believed to be relatively
small, because increases in SAH, the substrate for SAH-hydrolase, are
markedly less than the increases in 5'-AMP, the substrate for
5'-nucleotidase. In addition, we need to consider the activity of
adenosine kinase and adenosine deaminase as potential determinants of
adenosine production in rat cardiomyocytes.25 Schrader et
al28 reported that adenosine kinase influences the net
rate of adenosine production. Since we used 2'-deoxycoformysin and
5'-iodotubercidin to inhibit both enzymes and did not measure these two
enzyme activities directly, the present study does not determine
the role of activated protein kinase C in the modulation of the
activity of either adenosine kinase or deaminase.
Because we found
evidence that ecto-5'-nucleotidase plays an important
role in adenosine production from cardiomyocytes, we needed to examine
whether extracellular 5'-AMP is present around cardiomyocytes. We
showed that 5'-AMP can be detected in the medium in which the
cardiomyocytes are suspended and that hypoxia increases the amount of
5'-AMP. Even in the hypoxic condition, since most of the cardiomyocytes
were intact, the detection of 5'-AMP may not be a reflection of
cellular damages. Indeed, Bunger31 also reported that a
compartment of 5'-AMP in the interstitial space exists in hearts.
Furthermore, it is reported that 5'-AMP can be released from cells and
is converted to adenosine outside the cells.32 33
Thus, we
must consider the changes in the amount of 5'-AMP to be the cause of
increased adenosine release in the norepinephrine-,
methoxamine-, and PMA-treated conditions, because increases in
adenosine release through ecto-5'-nucleotidase may simply be
attributable to increased accumulation of the substrate, 5'-AMP.
However, Table 2 shows that 5'-AMP does not increase in
these groups,
indicating that changes in 5'-AMP are not the cause of increased
adenosine release.
The present results revealed that
1-adrenoceptor
activation is an important regulator for activation of
ecto-5'-nucleotidase. 1-Adrenoceptor activation is known
to result in activation of protein kinase C and production of inositol
1,4,5-triphosphate (IP3), which may modulate the activity
of ecto-5'-nucleotidase. The present study revealed that activation
of protein kinase C is critically important, rather than the production
of IP3, because (1) activation of
ecto-5'-nucleotidase due to 1-adrenoceptor stimulation
is prevented by GF109203X, an inhibitor of protein kinase C; (2) PMA
activates ecto-5'-nucleotidase; (3) the time courses of protein kinase
C activation precede the time courses of activation of
ecto-5'-nucleotidase in the groups of norepinephrine, methoxamine, and
PMA treatments; and (4) the time courses of the activation of
ecto-5'-nucleotidase in the norepinephrine-, methoxamine-,
and PMA-treated groups are comparable. As shown in Table 1,
protein
kinase C in the membrane fraction was almost dependent both on
Ca2+ and phospholipid, suggesting that protein
kinase C (PKC) activated by norepinephrine, methoxamine, and PMA was
conventional PKC (cPKC). The kinase activity independent of
Ca2+ and phospholipid in Table 1 may be
attributable
to protein kinase C other than cPKC, such as novel PKC (nPKC) or other
type of protein kinase. Although we depleted phospholipid in the assay
system to test the phospholipid dependency of kinase activity, the
contamination of phospholipid of the membrane fraction cannot be
excluded, which may partially account for the presence of
phospholipid-independent kinase.
The subcellular mechanism by which protein kinase C activates ecto-5'-nucleotidase is an interesting issue, although we could not clarify the exact mechanisms to explain our observation. One possibility is that activation of protein kinase C changes the cellular membrane characteristics around ecto-5'-nucleotidase and inhibits the internalization of this enzyme. Another possibility is that activation of protein kinase C may phosphorylate and activate the proteins that regulate activation of 5'-nucleotidase. Allosteric factors related to 5'-nucleotidase may be partially responsible for the activation of this enzyme, and activation of protein kinase C may modify this allosteric effect. Although these hypotheses are plausible, no data to support or refute any one of these theories were generated from the present study.
Pathophysiological Relevance in the Heart
Myocardial ischemia
has been reported to release norepinephrine
from the presynaptic vesicles,13 which activates
1-adrenoceptors and thereby protein kinase C.
Furthermore, ischemia translocates protein kinase C from the cytoplasm
to the membrane,14 which means the activation of protein
kinase C. In this situation, ATP and ADP are coreleased with
norepinephrine from the presynaptic vesicles. Released ATP and ADP can
be converted to 5'-AMP, which becomes the substrate for adenosine.
Therefore, the phenomenon of this 1-adrenoceptor
activation due to released norepinephrine increasing
ecto-5'-nucleotidase activity may effectively convert 5'-AMP to
adenosine. Importantly, 1-adrenoceptor–mediated
increases in adenosine release may contribute to
cardioprotection.3 15 16 Several lines of
evidence support
the proposition that adenosine is an important factor for
cardioprotection against ischemic and reperfusion
injury1 2 3 and that
1-adrenoceptor–mediated
increases in ecto-5'-nucleotidase activity and release of adenosine
improve reperfusion injury.3 15 16
The ultimate goal of cardioprotection is to limit infarct size in acute myocardial infarction. Administration of exogenous adenosine has been shown to limit infarct size, and adenosine mediates the infarct size–limiting effects of ischemic preconditioning.34 As a possible mechanism, we have shown that ischemic preconditioning is mediated by activation of ecto-5'-nucleotidase,35 36 37 which can be mediated by activation of protein kinase C during brief periods of ischemia.14 Thus, the present observation hints that the linkage between protein kinase C, ecto-5'-nucleotidase, and cardioprotection can be a clinical strategy to protect myocardium against ischemia and reperfusion injury.
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Acknowledgments |
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This work was supported by a Grant-in-Aid for Scientific Research (03670449) from the Ministry of Education, Science, and Culture, Japan, and by a grant from the Uehara Memorial Foundation, Japan. The authors are grateful to Noriko Tamai, Yoshitomo Edahiro, and Shinya Suzuki for their technical assistance.
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Footnotes |
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Reprint requests to Masafumi Kitakaze, MD, PhD, The First Department of Medicine, Osaka University School of Medicine, 2-2 Yamadaoka, Suita 565, Japan.
Received August 22, 1994; accepted November 26, 1994.
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References |
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