0022-3565/01/2981-71–76$3.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics
JPET 298:71–76, 2001
Vol. 298, No. 1
3630/911046
Printed in U.S.A.
Histamine Increases Interstitial Adenosine Concentration via
Activation of Ecto-5⬘-nucleotidase in Rat Hearts in Vivo
TOSHIO OBATA, SHUNICHIRO KUBOTA, and YASUMITSU YAMANAKA
Department of Pharmacology, Oita Medical University, Hasama-machi, Oita, Japan (T.O., Y.Y.); and Department of Physiological Chemistry and
Metabolism, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan (S.K.)
Received December 6, 2000; accepted March 14, 2001
This paper is available online at http://jpet.aspetjournals.org
Histamine induces catecholamine release in several organ
systems (Flacke et al., 1967; Albinus and Sewing, 1973;
Marco et al., 1980). Myocardial ischemia is associated with
an enhanced release of norepinephrine (Imamura et al.,
1994, 1996; Obata et al., 1994). Adenosine, an endogenous
nucleoside, is an important biochemical intermediate in cellular metabolism and has cardioprotective effects in myocardial ischemia (Lasley et al., 1990; Ely and Berne, 1992; Lasley and Mentzer, 1992; Thornton et al., 1992). Although the
interaction between histamine and adenosine in the rat heart
is unclear, recent reports have demonstrated the interaction
between central histaminergic and adrenergic systems in
cardiovascular response (Bealer, 1993; Bealer and Abell,
1994). Some investigators (Kitakaze et al., 1995; Sato et al.,
1997) reported that enhanced activation of protein kinase C
(PKC) increased 5⬘-nucleotidase activity, leading to an increased release of adenosine, in isolated rat cardiomyocytes.
Furthermore, in isolated rat cardiomyocytes, the activation
of 5⬘-nucleotidase was shown to be mediated by the activation of PKC (Kitakaze et al., 1995). Adenosine exerts multiple
actions throughout the body and modifies various cardiovasThis study was supported by Grants-in-Aid for Scientific Research from the
Ministry of Education, Science, Sports, and Culture, and Health Science Research Grants for Research on Environmental Health from the Ministry of
Health and Welfare, Japan.
concentration. Accumulation of norepinephrine in the extracellular fluid elicited by pargyline (100 ␮M), a monoamine oxidase
inhibitor, significantly increased histamine-induced adenosine
production. Okadaic acid (50 ␮M), an inhibitor of protein phosphatase, enhanced the histamine-induced increase in adenosine concentration. Norepinephrine is known to activate ␣1adrenoceptors and PKC. Taken together, the results
demonstrate that histamine-released norepinephrine activates
both ␣1-adrenoceptors and PKC, which increased ecto-5⬘-nucleotidase activity and augmented release of adenosine in rat
hearts.
cular functions (Berne, 1980). The formation and release of
adenosine in the ischemic myocardium is enhanced, and the
adenosine is derived from the enzymatic dephosphorylation
of adenosine 5⬘-monophosphate (AMP) by 5⬘-nucleotidase
(Frick and Lowenstein, 1976; Thornton et al., 1992). It is
suggested that, in dog hearts, stimulation of ␣1-adrenoceptor
augments adenosine production during ischemia by enhancing 5⬘-nucleotidase activity, which can limit the size of the
infarct (Kitakaze et al., 1994). The present study was undertaken to clarify whether histamine affects the norepinephrine-mediated interstitial adenosine production.
To achieve this goal, we measured the concentration of
interstitial adenosine in in vivo hearts using a flexibly
mounted microdialysis technique that we developed (Obata
et al., 1994, 1998). The production of adenosine under normoxic conditions is attributed primarily to the transmethylation of S-adenosylhomocysteine (SAH) catalyzed by SAH
hydrase; the hydrolysis of AMP by ecto-5⬘-nucleotidase, the
main pathway for adenosine production under ischemic conditions, is considered to be minimal (Sparks and Bardenheuer, 1986; Liu et al., 1991). To mimic ischemic conditions,
we measured the concentration of dialysate adenosine under
continuous supply of AMP, the substrate for 5⬘-nucleotidase,
through the microdialysis probe. Using this system, we have
reported that the level of AMP-primed dialysate adenosine
ABBREVIATIONS: PKC, protein kinase C; ␣,␤-meADP, ␣,␤-methyleneadenosine 5⬘-diphosphate; SAH, S-adenosylhomocysteine; HPLC, highperformance liquid chromatography.
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ABSTRACT
We examined whether histamine enhances the production of
interstitial adenosine via stimulation of ecto-5⬘-nucleotidase (a
key enzyme responsible for adenosine production) using microdialysis techniques in in situ rat hearts. The microdialysis
probe was implanted in the left ventricular myocardium of
anesthetized rats and perfused in the presence of adenosine
5⬘-monophosphate (AMP). Histamine (10 –500 ␮M) administered into the perfusate had a tendency to increase the adenosine concentration. In the presence of prazosin (50 ␮M), an
antagonist of ␣1-adrenoceptors, or of chelerythrine (10 ␮M), a
protein kinase C (PKC) inhibitor, and in reserpinized rats, histamine failed to increase the AMP-primed dialysate adenosine
72
Obata et al.
reflects the activity of ecto-5⬘-nucleotidase in the particular
site of the interstitial space of the myocardium (Obata and
Yamanaka, 2000). The results in the present study demonstrate that histamine increased the production of interstitial
adenosine via norepinephrine-mediated activation of ecto-5⬘nucleotidase.
Materials and Methods
Animal Preparation
Microdialysis Technique
Details of the technique required for manipulation of the flexibly
mounted microdialysis probe in in vivo rat hearts (to measure the
interstitial adenosine) have been described previously (Obata et al.,
1994). In brief, the tip of the microdialysis probe (3 mm in length and
220-␮m o.d. with the distal end closed) was made of dialysis membrane (cellulose membrane 10 ␮m thick with a 50,000 molecular
weight cut-off). Two fine silica tubes (75-␮m i.d.) were inserted into
the tip of a cylinder-shaped dialysis probe and served as an inlet for
the perfusate and an outlet for the dialysate, respectively. The inlet
tube was connected to a microinjection pump (CMA/100, Carnegie
Medicine, Stockholm, Sweden), and the outlet tube led to the dialysate reservoir. These tubes were supported loosely at the midpoint on
a semirotatable stainless steel wire so that their movement fully
synchronized with the rapid up-and-down motion of the tip caused by
the heart beats. The probe was implanted from the epicardial surface
into the left ventricular myocardium and was perfused through the
inlet tube with Tyrode’s solution of the following composition (in
mM): NaCl, l37; KCl, 5.4; CaC12, 1.8; MgC12, 0.5; NaH2P04, 0.16;
NaHCO3, 3.0; glucose, 5.5; and HEPES, 5.0 (pH ⫽ 7.4 adjusted with
NaOH). The Tyrode’s solution that flowed out of the cut end of the
inlet tube entered the extracellular space across the dialysate membrane by diffusion. The interstitial fluid diffused back into the cavity
of the probe, and the dialysate left the probe through the orifice of the
outlet tube. The perfusion rate was 1.0 ␮l/min. The relative recovery
of adenosine measured using this flow rate (1.0 ␮l/min) was 18.0 ⫾
1.6% (n ⫽ 6).
Analytical Procedure
Measurements of Adenosine Concentration in Dialysate.
The dialysate was collected (at the rate of 1.0 ␮l/min) into a series of
wells for every 15 min consecutively (15 ␮l in each well). A 10-␮l
aliquot of the dialysate sample was used for the detection of adenosine, and we measured the concentration using reversed-phase
high-performance liquid chromatography (HPLC). Separation of the
compounds was achieved on Eicompak MA-5 ODS columns (5 ␮m,
4.6 ⫻ 150 mm; Eicom, Kyoto, Japan) with the mobile phase consisting of 200 mM KH2PO4 (pH ⫽ 3.8 adjusted with phosphoric acid) and
5% (v/v) acetonitrile. The flow rate was set at 1.0 ␮l/min using a
pumping system (PU-980, JASCO Corp., Tokyo, Japan). The absorbance of the column eluate was monitored at 260 nm using an
ultraviolet detector (UV-970, JASCO Corp.). The absorbance peak of
adenosine was quantified by comparing the retention time and peak
height with a known adenosine standard at concentrations of 1 and
10 ␮M. Concentrations of adenosine are expressed as a raw value
Experimental Protocol
We measured the time-dependent changes of the dialysate adenosine concentration in the presence of AMP (AMP-primed dialysate
adenosine concentration) and evaluated the activity of ecto-5⬘-nucleotidase. Under a constant supply of AMP, the dialysate adenosine is
considered to originate from enzymatic dephosphorylation of AMP by
endogenous ecto-5⬘-nucleotidase since ␣,␤-methyleneadenosine 5⬘diphosphate (␣,␤-meADP, 100 ␮M), an inhibitor of ecto-5⬘-nucleotidase, completely inhibited the AMP-primed dialysate adenosine
(Obata and Yamanaka, 2000). Therefore, the level of dialysate adenosine measured in the presence of AMP is an appropriate measure of
the activity of ecto-5⬘-nucleotidase in rat hearts in situ. In this series
of experiments, AMP at a concentration of 100 ␮M was perfused
throughout the experiment via the probe, and the dialysate sampling
was started after a 30-min equilibration period.
Drugs Used
Histamine hydrochloride and AMP (Wako Pure Chemical Co.,
Osaka, Japan) and pargyline hydrochloride (Sigma, St. Louis, MO,
and Osaka, Japan) were dissolved with the Tyrode’s solution just
before the start of experiments to acquire the desired final concentrations, as given in the text. ␣,␤-meADP (Sigma) and chelerythrine
(Sigma) were dissolved in distilled water and kept as 10 mM stock
solutions. Okadaic acid (a kind gift from Fugisawa Pharmaceutical
Co., Osaka, Japan) was dissolved in dimethyl sulfoxide as a 10 mM
stock solution. An appropriate volume of these stock solutions was
added to Tyrode’s solution just before use, as indicated under Results. Reserpine was purchased from Daiichi Pharmaceutical Co.
(Tokyo, Japan).
Statistical Analysis
All values are expressed as means ⫾ S.E.M. The significance of
difference was determined by using ANOVA with Fisher’s post hoc
test. A P value of less than 0.05 was considered to be statistically
significant.
Results
The Effect of Histamine on Adenosine Formation. We
first examined the effect of histamine on the dialysate aden-
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The study was performed with Wistar rats of either sex, weighing
300 to 400 g, that were anesthetized by an intraperitoneal injection
of chloral hydrate (400 mg/kg). After intubation, the rat was mechanically ventilated with room air supplemented with oxygen. The chest
was opened at the left 5th intercostal space, and the pericardium was
removed to expose the left ventricle. In the case of reserpinized rats,
reserpine (5 mg/kg) was injected intravenously 24 h before the experiment. All procedures in dealing with the experimental animals
met the guideline principles stipulated by the Physiological Society
of Japan and the Animal Ethics Committee of the Oita Medical
University.
unless otherwise indicated. The limit of the assay for adenosine is
0.42 ␮M.
Measurements of Norepinephrine Concentration in Dialysate. To determine the level of norepinephrine, the heart was perfused with Ringer’s solution consisting of 147 mM NaCl, 2.3 mM
CaCl2, and 4 mM KCl (pH 7.4) (Obata et al., 1994; Yamazaki et al.,
1997). Norepinephrine assay was performed using HPLC with an
electrochemical procedure. To make the standard norepinephrine
solution, norepinephrine was dissolved in the Ringer’s solution.
When the perfusion rate of 1.0 ␮l/min was used, the relative recovery, using the standard norepinephrine solution (1 ␮M), was l7.0 ⫾
0.7%. The dialysate samples were collected into a small collecting
tube containing 15 ␮l of 0.1 N HClO4 for every 15 min consecutively
for the adenosine measurements. The samples were immediately
injected into an HPLC-electrochemical system equipped with a
glassy carbon working electrode (Eicom, Kyoto, Japan) and an analytic reverse-phase column on an Eicompak MA-5ODS column (5 ␮m,
4.6 ⫻ 150 mm; Eicom). The working electrode was set at a detector
potential of 0.75 V. Each liter of mobile phase contained 1.5 g of
1-heptanesulfonic acid sodium salt, 0.1 g of Na2EDTA, 3 ml of triethylamine, and 125 ml of acetonitrile. The pH of the solution was
adjusted to 2.8 with 3 ml of phosphoric acid. When dialysate norepinephrine levels reached a steady state at 120 min after probe implantation, histamine was directly infused in rat heart through a
microdialysis probe. The limit of the assay for norepinephrine is
0.005 ␮M.
Ecto-5ⴕ-nucleotidase Activation by Histamine
Fig. 1. Effect of histamine on the production of interstitial adenosine in
rat ventricular myocardium. A, sequential changes of the dialysate adenosine concentration measured in the presence of 100 ␮M AMP throughout. Histamine (100 ␮M) was added to the perfusate for 45 min, as
indicated by a horizontal bar (n ⫽ 6). B, effect of ␣,␤-meADP (100 ␮M) on
histamine-induced increases in dialysate adenosine (n ⫽ 6). The abscissa
denotes the time in minutes before and after the introduction of histamine. Values are means ⫾ S.E.M. *P ⬍ 0.05, **P ⬍ 0.001 versus predrug
value.
Fig. 2. Concentration-dependent effect of histamine on the AMP-primed
(100 ␮M) dialysate adenosine. The ordinate scale indicates concentrations of dialysate adenosine, each measured 30 to 45 min after application
of various concentrations of histamine (10, 50, 100, and 500 ␮M), and is
shown as a percentage relative to the value measured just before histamine was applied (100%). Values are means ⫾ S.E.M. (n ⫽ 6). ns,
nonsignificant. *P ⬍ 0.05 versus predrug value (n ⫽ 6 in each column).
Fig. 3. Effects of histamine on the norepinephrine concentration in the
dialysate. The histamine was introduced in the perfusate in intact (A)
and reserpinized rat hearts (B), as indicated by horizontal bars. The
abscissas show the time after implantation of the dialysis probe. Values
are means ⫾ S.E.M. (n ⫽ 6). *P ⬍ 0.05 versus predrug value (n ⫽ 6 in
both A and B).
dialysate were the result of increased PKC activity via ␣1adrenoceptor stimulation. To clarify this, we used prazosin,
an ␣1-adrenoceptor antagonist, or chelerythrine, a potent
and selective PKC inhibitor, that interacts with the catalytic
domain of this enzyme (Herbert et al., 1990). In the presence
of prazosin (50 ␮M), histamine (100 ␮M) failed to increase
the dialysate adenosine (Fig. 4A). In contrast, atenolol, a
␤1-adrenoceptor antagonist, did not prevent the histamineinduced increase in dialysate adenosine even in the presence
of a high concentration of atenolol (50 ␮M); histamine (100
␮M) significantly increased the dialysate adenosine concentration (by 41.3 ⫾ 13.8%, P ⬍ 0.05, not illustrated). On the
other hand, in the presence of chelerythrine (10 ␮M), histamine did not increase the dialysate adenosine (Fig. 4B). In
addition, in reserpine-treated animals, histamine did not
significantly increase the level of adenosine in the dialysate
(from 3.47 ⫾ 0.63 to 3.41 ⫾ 0.58 ␮M) (Fig. 4C). These results
suggest that histamine-released endogenous norepinephrine
increased the AMP-primed dialysate adenosine concentration (i.e., the activity of ecto-5⬘-nucleotidase) via activation of
PKC. To further support this notion, we examined the effects
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osine concentration in the presence of AMP and evaluated
the activity of ecto-5⬘-nucleotidase in vivo. In this series of
experiments, AMP was perfused throughout the experiments
via a microdialysis probe. The dialysate sampling was
started after a 30-min equilibration period, as described previously (Sato et al., 1997). After obtaining two control fractions (a dialysate of 30 – 45 and 45– 60 min), histamine (100
␮M) was introduced through the probe in the presence of
AMP. The baseline level of dialysate adenosine measured in
the absence of exogenous AMP was ⬃0.5 ␮M, which was ⬃18
times lower than the level of dialysate adenosine observed in
the presence of 100 ␮M AMP (⬃9 ␮M). Histamine (100 ␮M)
significantly increased the level of dialysate adenosine from
8.26 ⫾ 0.66 to 11.68 ⫾ 1.09 ␮M at 30 to 45 min after histamine was applied (n ⫽ 6, P ⬍ 0.05). After the removal of
histamine from the perfusate, the adenosine concentration
was decreased to 7.68 ⫾ 0.95 ␮M in 30 min (Fig. 1A). In
contrast, the introduction of ␣,␤-meADP (100 ␮M) significantly decreased the concentration of adenosine (0.51 ⫾ 0.18
␮M) within 45 min (a dialysate of 90 –105 min) (n ⫽ 6, P ⬍
0.05). After removal of these drugs from the perfusate, the
concentration of dialysate adenosine was gradually restored
and reached a level of 10.50 ⫾ 1.85 ␮M (a dialysate of 135–
150 min) (Fig. 1B). Similar experiments were repeated using
various concentrations of histamine (10, 50, 100, and 500
␮M), and the results are summarized in Fig. 2. Histamine
had a tendency to increase the level of AMP-primed dialysate
adenosine over the concentration range of 10 to 500 ␮M; the
maximum effect, 143.8 ⫾ 15.4% of control (n ⫽ 6, P ⬍ 0.05),
was obtained at 100 ␮M histamine.
The Effect of Histamine on Norepinephrine Release.
After obtaining two control fractions (a dialysate of 30 – 45
and 45– 60 min), histamine (100 ␮M) was introduced through
the probe in the presence of 100 ␮M AMP. As shown in Fig.
3A, histamine (100 ␮M) significantly increased the level of
norepinephrine in the dialysate (n ⫽ 6, P ⬍ 0.05). In contrast,
in rats treated with reserpine (see Materials and Methods),
the levels of norepinephrine remained suppressed (n ⫽ 6)
(Fig. 3B).
The Role of ␣1-Adrenoceptors and PKC in the Histamine-Induced Adenosine Formation. We examined
whether the histamine-induced increases of adenosine in
73
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Obata et al.
Fig. 4. Effects of prazosin and chelerythrine on the dialysate adenosine
concentration. The effect of histamine
(100 ␮M) on the adenosine concentration was studied in the presence of 50
␮M prazosin (n ⫽ 5, A) or 10 ␮M chelerythrine (n ⫽ 6, B) in intact rat heart
or in reserpinized rat heart (n ⫽ 5, C).
Values are means ⫾ S.E.M.
Discussion
The conversion of AMP to adenosine by 5⬘-nucleotidase
may be a crucial step for cardioprotection during myocardial
Fig. 5. Effect of high concentration of pargyline on the dialysate adenosine concentration under histamine in the perfusate. The abscissa denotes the time in minutes after the introduction of histamine. Pargyline
(100 ␮M) was added to the perfusate for 45 min, as indicated by a
horizontal bar (n ⫽ 6). Values are means ⫾ S.E.M. *P ⬍ 0.05, versus
predrug value.
ischemia. We assessed the activity of ecto-5⬘-nucleotidase (a
key enzyme responsible for adenosine production) and examined the effects of histamine on the production of interstitial
adenosine using a flexibly mounted microdialysis technique
(Obata et al., 1994, 1998). We provided the evidence that the
level of dialysate adenosine measured in the presence of AMP
reflects the activity of ecto-5⬘-nucleotidase in the particular
tissue (Obata and Yamanaka, 2000). In the present study, we
have demonstrated that histamine enhanced the production
of interstitial fluid adenosine produced via stimulation of
ecto-5⬘-nucleotidase in rat hearts using microdialysis technique.
AMP-induced increases in the dialysate adenosine concentration was shown to be dependent on the AMP concentrations used, and the EC50 of AMP was ⬃100 ␮M (Sato et al.,
1997), a value close to the Km (Michaelis constant) estimated
for ecto- (rather than for cytosolic) 5⬘-nucleotidase in rat
hearts (Sullivan and Alpers, 1971). The baseline level of
dialysate adenosine was ⬃0.5 ␮M. Based on the recovery rate
of tissue adenosine (18%), the concentration of adenosine in
the interstitial fluid of the ventricular muscle located adjacent to the dialysis membrane was ⬃2.8 ␮M, a value comparable to that reported in other studies, i.e., 0.3 to 3.6 ␮M,
using a conventional microdialysis technique (Van Wylen et
al., 1990, 1992) or a porous nylon sampling disc technique
(Zhu et al., 1991). Thus, the value of 2.8 ␮M observed in the
Fig. 6. Effect of okadaic acid on the dialysate adenosine concentration
under histamine in the perfusate. Okadaic acid (50 ␮M) was added to the
perfusate for 45 min, as indicated by a horizontal bar (n ⫽ 6). Values are
means ⫾ S.E.M. *P ⬍ 0.05, versus predrug value.
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of pargyline, a monoamine oxidase inhibitor, on the production of interstitial adenosine. In the presence of a high concentration of pargyline (100 ␮M), histamine (100 ␮M) significantly increased the dialysate adenosine from 11.34 ⫾ 0.96
to 14.16 ⫾ 0.70 ␮M at 30 to 45 min after pargyline was
applied (n ⫽ 6, P ⬍ 0.05) (Fig. 5).
If the activity of ecto-5⬘-nucleotidase were increased by
PKC as suggested above, phosphorylation of the enzyme
could be responsible for this increase. To test this possibility,
we used okadaic acid, a protein phosphatase inhibitor (Bialojan and Takai, 1988). Okadaic acid (50 ␮M) per se did not
affect the dialysate adenosine level measured in the presence
of AMP (100 ␮M, n ⫽ 5, not illustrated). However, when
okadaic acid (50 ␮M) was introduced in the presence of histamine (100 ␮M), the AMP-primed dialysate adenosine level
significantly increased from 11.50 ⫾ 0.61 to 15.36 ⫾ 1.24 ␮M
at 30 to 45 min after okadaic acid was introduced (n ⫽ 6, P ⬍
0.05) (Fig. 6). These results support the notion that phosphorylation of ecto-5⬘-nucleotidase by PKC augmented the
activity of this enzyme.
Ecto-5ⴕ-nucleotidase Activation by Histamine
of adenosine catalyzed by an enhanced activity of ecto-5⬘nucleotidase in the rat heart in vivo. We previously reported
that diacylglycerol, a potent PKC activator (Nishizuka,
1995), increased the AMP-primed dialysate adenosine (Sato
et al., 1998). It is known that norepinephrine stimulates
␣1-adrenoceptors and leads to activation of PKC (Fedida et
al., 1993). During acute regional ischemia, the interstitial
concentration of norepinephrine in the ischemia region is
reported to be increased (Schömig, 1989). Taken together, the
results suggest that histamine-released norepinephrine
stimulated ␣1-adrenoceptors and activated PKC, leading to
activation of ecto-5⬘-nucleotidase and release of adenosine.
We examined the effect of pargyline, a monoamine oxidase
inhibitor, on the production of interstitial adenosine. In the
presence of histamine, pargyline (100 ␮M) significantly increased the dialysate adenosine (Fig. 5). The results indicate
that accumulation of norepinephrine in the extracellular
fluid elicited by pargyline led to the production of adenosine.
Our results indicate that histamine-released norepinephrine
elevates adenosine by 5⬘-nucleotidase activation. Specifically, it has been shown that ␣1-adrenoceptor stimulation
and subsequent PKC activation is apparently one of the
pathways that causes an adenosine rise through 5⬘-nucleotidase activation (Sato et al., 1997).
The steady-state production of adenosine, i.e., the steadystate concentration of dialysate adenosine, may depend on
the equilibrium between phosphorylation and dephosphorylation of ecto-5⬘-nucleotidase. Okadaic acid enhances phosphorylation by inhibiting protein phosphatases (Bialojan and
Takai, 1988). Our observation that okadaic acid enhanced
the effect of histamine on the production of adenosine (Fig. 6)
suggests that PKC phosphorylated ecto-5⬘-nucleotidase and
increased its enzyme activity, leading to increased production of adenosine. As shown in Fig. 4, the adenosine level in
reserpinized rat heart was about half that of nontreated
control. Although the exact mechanism of reduced adenosine
level in reserpinized rats is unclear, it may be explained as
follows. Komachi et al. (1993, 1994) reported that membraneassociated immunoreactive protein kinase C was reduced in
reserpinized rat brain. The change of PKC distribution may
lead to decreased PKC activity and decreased phosphorylation of ecto-5⬘-nucleotidase, and as a consequence, decreased
ecto-5⬘-nucleotidase activity would reduce the adenosine
level. Ischemia activates PKC via an ␣1-adrenoceptor-dependent and -independent mechanism. The latter mechanism of
activation was secondary to translocation of PKC from the
cytosol to the sarcolemma of cardiac muscles: the translocated PKC may then activate ecto-5⬘-nucleotidase, perhaps
via modification of some of the latter enzyme from inside of
the membrane, and as a consequence, interstitial adenosine
would increase. Moreover, several lines of experimental evidence suggest that stimulation of a variety of G proteincoupled receptors (e.g., adenosine, A1, ␣1-adrenergic, muscarine, bradykinin, and endothelin-1 receptors) leads to the
activation of PKC (Cohen and Downey, 1996). Although the
contribution of histamine to this phenomenon is less known,
it is possible that histamine, a catecholamine releaser, may
contribute to ischemic preconditioning. However, further research is necessary to confirm the relation between histamine release and ischemic preconditioning. The present
study provides in vivo evidence as follows: histamine-released norepinephrine stimulated an ␣1-adrenoceptor-depen-
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present study is within the range obtained in previous studies. The AMP-induced adenosine was most probably generated by enzymatic dephosphorylation of AMP by ecto-5⬘nucleotidase, and the baseline production of adenosine was
probably derived from hydrolysis of SAH. In the presence of
the selective inhibitor of ecto-5⬘-nucleotidase, ␣,␤-meADP
(Hori and Kitakaze, 1991), at a concentration of 100 ␮M in
the perfusate, AMP (100 ␮M)-induced increases of adenosine
in the dialysate were completely inhibited and remained at
⬃0.51 ␮M, a level close to the baseline. Therefore, it is
reasonably assumed that the level of dialysate adenosine is
proportional to the adenosine concentration in the interstitial space of the myocardium and reflects the ecto-5⬘-nucleotidase activity in this tissue. The rationale and the relevance of this method were addressed in our previous reports
(Sato et al., 1997; Obata and Yamanaka, 2000). In the
present experiment, the administration of drugs was through
the microdialysis probe. Although the exact mechanism by
which histamine induced adenosine production is unclear,
histamine clearly increased the level of adenosine in the rat
heart (Fig. 1A). The introduction of ␣,␤-meADP in the presence of histamine significantly decreased the level of AMPprimed dialysate adenosine (Fig. 1B). Our preliminary observation showed that histamine not only increased the
concentration of adenosine in the dialysate but also that of
inosine, and in the presence of ␣,␤-meADP, histamine decreased the levels of both adenosine and inosine. Therefore, it
is unlikely that the reduction of interstitial adenosine concentration by histamine in the presence of ␣,␤-meADP was
due to increased activity of adenosine deaminase. Taken
together, it is likely that the histamine-induced increase of
adenosine was due to the activation of ecto-5⬘-nucleotidase.
However, we cannot rule out other possibilities. For example,
histamine attenuated the breakdown of adenosine by inhibiting adenosine deaminase, leading to the increase in dialysate adenosine. However, histamine (100 ␮M) did not affect
the level of dialysate adenosine when measured in the absence of AMP (T. Obata, unpublished observation). Therefore, it is not likely that histamine attenuated the breakdown
of adenosine. Namely, the effective concentrations outside
the dialysis membrane are probably lower than the dialysate
concentration. AMP supplied from an inlet tube diffused out
into the interstitial fluid through the dialysis membrane and
was converted to adenosine by endogenous 5⬘-nucleotidase.
We examined the effect of ␣,␤-meADP, a selective inhibitor of
ecto-5⬘-nucleotidase, which was unable to access to cytosolic
5⬘-nucleotidase because it cannot penetrate the sarcolemma
of heart muscle cells (Headrick et al., 1992). Since ␣,␤meADP completely inhibited histamine-induced increases in
dialysate adenosine concentrations without affecting the
baseline level of adenosine, the AMP-induced increase in the
adenosine concentration was most probably derived from
enzymatic dephosphorylation of AMP by ecto-5⬘-nucleotidase, and the baseline production of adenosine was probably
derived from hydrolysis of SAH.
Histamine is released during myocardial infarction and
ischemic arrhythmias (Masini et al., 1987). We demonstrated
that histamine increased the adenosine concentration measured in the presence of 100 ␮M AMP, which was inhibited
by ␣1-antagonist (prazosin) (Fig. 4A) or PKC inhibitor (chelerythrine, 10 ␮M) (Fig. 4B). Thus, we have shown a clear and
important link between activation of PKC and the production
75
76
Obata et al.
dent and -independent mechanism. PKC phosphorylated
ecto-5⬘-nucleotidase and enhanced its enzyme activity, leading to the increased production of adenosine in rat hearts.
Estimation of ecto-5⬘-nucleotidase activity by using flexibly
mounted microdialysis probes perfused with AMP may be
useful in future studies to elucidate the regulatory influences
of ecto-5⬘-nucleotidase on the production of adenosine.
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Address correspondence to: Dr. Toshio Obata, Department of Pharmacology, Oita Medical University, Hasami-machi, Oita 879-5593 Japan. E-mail:
[email protected]
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