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Description  |
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FIELD OF THE INVENTION
This invention relates to methods of cardiac treatment.
BACKGROUND OF THE INVENTION
The binding of catecholamines, e.g., epinephrine or norepinephrine, to
.beta.-adrenergic cell surface receptors ("adrenoceptors") in the heart
increases cardiac contractile and metabolic activity. The
.beta.-adrenoceptor-mediated cardiac stimulation involves a chain of
events that includes binding of the catecholamine to the receptor,
adenylyl cyclase activation, increased adenosine 3',5'-monophosphate
(cAMP) formation, protein kinase activation, and action by phosphorylated
proteins. In the heart, adenosine counteracts the .beta.-adrenergic
stimulation of adenylyl cyclase activity, thereby counteracting
.beta.-adrenoceptor-mediated cardiac stimulation. These negative
regulatory effects of adenosine have been called the "antiadrenergic"
actions of adenosine. (They have also been called the "indirect
inhibitory" and "retaliatory" actions of adenosine.)
The antiadrenergic actions of adenosine on the heart are mediated by type
A.sub.1 adenosine receptors, which, like .beta.-adrenergic receptors, are
cell surface receptors. Type A.sub.1 adenosine receptors have a high
affinity for adenosine. Accordingly, the antiadrenergic actions of
adenosine in the heart occur at relative low interstitial (i.e.
extracellular) adenosine concentrations, i.e., 0.1-10 .mu.M. Adenosine
administered to the heart, at these low concentrations, in the absence of
.beta.-adrenergic stimulation, has no detectable direct effect on the
above-mentioned metabolic and mechanical parameters.
A second type of cell surface receptor for interstitial adenosine,
designated A.sub.2, has a lower affinity for adenosine, i.e.,
approximately two to three orders of magnitude lower than the adenosine
affinity of A.sub.1 receptors. Type A.sub.2 adenosine receptors exist in
various mammalian tissues and have been detected in mammalian ventricular
myocytes.
SUMMARY OF THE INVENTION
We have discovered that activating adenosine A.sub.2 receptors in a
mechanically compromised mammalian heart causes an increase in the
contractile performance of the heart.
Accordingly, the invention features a method for increasing the contractile
performance of a compromised myocardium in a mammal, comprising
administering a therapeutically effective amount of an adenosine A.sub.2
receptor agonist to said mammal. The adenosine receptor agonist used in
the practice of this invention may be adenosine or any other compound that
binds specifically to adenosine receptors, including A.sub.2 receptors,
and thereby causes the signal transduction normally caused by the binding
of adenosine to the receptors. If the adenosine receptor agonist used is a
compound other than adenosine, preferably it is a compound that is
selective for adenosine A.sub.2 receptors.
A second compound which potentiates the beneficial effect of adenosine may
be administered to a mammal, in conjunction with adenosine or an adenosine
receptor agonist. The beneficial effects of adenosine could be potentiated
in at least two ways: (1) by enhancing the interstitial concentration of
adenosine in the myocardium; and (2) inhibiting the A.sub.1
receptor-mediated antiadrenergic effects of adenosine.
One means whereby the second compound could enhance the interstitial
concentration of adenosine is inhibition of the clearance (i.e., removal)
of interstitial adenosine. More particularly, clearance of interstitial
adenosine is inhibited by administering an adenosine transport inhibitor
in conjunction with adenosine or an adenosine receptor agonist. Another
means whereby the second compound could enhance the interstitial
concentration of adenosine is the inhibition of adenosine metabolism.
The A.sub.1 receptor-mediated antiadrenergic effects of adenosine are
inhibited by administering an A.sub.1 receptor antagonist, i.e., a
compound that binds specifically to A.sub.1 receptors and "blocks" them
without causing signal transduction.
The positive inotropic response of a mammalian heart resulting from
adenosine A.sub.2 receptor activation is relatively small, as compared to
the .beta.-adrenergic receptor-mediated response resulting from
catecholamine stimulation. The moderate increase in the contractile
performance of a compromised mammalian heart achieved according to the
present invention advantageously avoids overstimulation and concomitant
heart failure, which may result from more potent inotropes.
As used herein, "adenosine A.sub.2 receptor agonist" means adenosine or any
other compound that binds specifically to adenosine receptors, including
A.sub.2 receptors, and thereby causes the A.sub.2 receptor-mediated signal
transduction normally caused by the binding of adenosine to the A.sub.2
receptors.
As used herein, "adenosine receptor antagonist" means a compound that binds
specifically to one or more types of adenosine receptor without causing
the signal transduction normally caused by the binding of adenosine to the
receptor(s). A particular compound may be an antagonist for adenosine
A.sub.1 receptors, A.sub.2 receptors, or both.
As used herein, "compromised myocardium" means a myocardium whose
mechanical performance, in terms of an accepted parameter, is at least 10%
below that which is normal for an individual of similar size and age, as
measured under resting conditions or exercise conditions.
As used herein, in the context of an isolated myocyte, "contractile
performance" means any of the following: length change or shortening
("LC"), duration of shortening ("DS"), time to peak shortening ("TPS"),
maximal rate of shortening (+dL/dt.sub.max), time to 75% relaxation
("TR"), or maximal rate of relengthening (-dL/dt.sub.max).
As used herein, in the context of an intact heart, "contractile
performance" means any of the following: stroke volume, left ventricular
pressure ("LVP") change, time to peak pressure, maximal rate of pressure
increase (+dP/dt.sub.max), time to 75% relaxation, or maximal rate of
pressure decrease (-dP/dt.sub.max).
As used herein, "hypoxic" means arterial blood oxygen saturation of 90% or
lower, with normal arterial blood oxygen saturation being 95%.
As used herein, "inotropic" means influencing the contractility of muscular
tissue.
As used herein, "ischemic" means having a blood flow at least 10% below
that which is normal for an individual of similar size and age, as
measured under resting conditions or exercise conditions. In an adult
human, normal resting blood flow is approximately 1 ml/min/gram of
myocardial mass. During exercise, blood flow typically rises to
approximately 3-6 ml/min/gram of myocardial mass.
As used herein, a "therapeutically effective amount" of adenosine or an
adenosine receptor agonist is an amount that causes an increase of at
least 10% in stroke volume, LVP, or maximal rate of pressure increase
(+dP/dt.sub.max) in the mammal to which it is administered.
Other features and advantages of this invention will be apparent from the
following description of the preferred embodiments thereof, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic diagram of the instruments used to obtain contractile
information from single rat heart ventricular myocytes. Myocytes were
continuously suffused in a myocyte chamber (C) from right to left with
suffusion solution. Platinum wire electrodes in the base of the chamber
were used to elicit myocyte contractions at 0.5 Hz. The bottom and top of
the chamber are glass, so that when mounted on an inverted microscope (M),
contractions could be visualized. A line scan camera (LSC) fixed to the
inverted microscope was used to record the shortening of single
ventricular myocytes associated with each contraction.
FIGS. 2A-2D are graphs, generated on a computer screen, illustrating the
raw data in pixels indicating edge detection (length) collected over 1 sec
(FIG. 2A), myocyte length vs. time (FIG. 2B), myocyte length change
(.DELTA.length or LC) vs. time (FIG. 2C) and + and -dL/dt vs. time (FIG.
2D) for a single rat ventricular myocyte contraction. Time and monocyte
length are in msec and microns (.mu.m), respectively.
FIG. 3 is a graph illustrating the effect of isoproterenol ("ISO"),
phenylisopropyladenosine ("PIA"), and 1,3-dipropyl-8-cyclopentylxanthine
("DPCPX") on ventricular myocyte contraction. Traces are length changes
associated with single contractile shortenings of a representative rat
ventricular myocyte upon the sequential addition every 8-10 min of 0.2
.mu.M ISO, ISO+2 .mu.M PIA, and ISO+PIA+0.2 .mu.M DPCPX.
FIG. 4 is a graph illustrating the effect of ISO and DPCPX on ventricular
myocyte contraction in the presence of PIA. Traces are length changes
associated with single contractile shortenings of a representative rat
ventricular myocyte in the presence of 1 .mu.M PIA with sequential
addition every 8-10 min of 0.1 .mu.M ISO, and ISO +0.1 .mu.M DPCPX.
FIG. 5 is a graph illustrating the effect of
2-p-(2-carboxy-ethyl)phenethyl-amino-5'-N-ethylcarboxamido-adenosine
("CGS-21680") on ventricular myocyte contraction. Traces are length
changes associated with single contractile shortenings of a representative
rat ventricular myocyte upon sequential addition of increasing
concentrations of CGS-21680 (0.1 to 10 .mu.M) every 10 min.
FIG. 6 is a graph comparing CGS-21680 and ISO elicited contractile
responses in a rat ventricular myocyte. Traces are length changes
associated with single contractile shortenings of a representative myocyte
exposed to 0.2 .mu.M CGS-21680 for 5 min., washed for 15 min in suffusion
solution containing no CGS-21680 then exposed to 0.2 .mu.M ISO for 3 min.
FIG. 7 is a graph illustrating the effect of CGS-21680 and
9-chloro-2-(2-furanyl)-5,6-dihydro-1,2,4-triazolo-(1,5-C)quinazolin-5-imin
e ("CGS-15943") on ventricular myocyte contraction. Traces are length
changes associated with single contractile shortenings of a representative
rat ventricular myocyte upon the sequential addition of 10 .mu.M CGS-21680
and then 8-10 min later CGS-21680+10 .mu.M CGS-15943.
FIG. 8 is a graph illustrating the effect of PIA on ISO stimulated
ventricular myocyte membrane adenylyl cyclase activity. Adenylyl cyclase
activity was assessed in the absence (basal, B) or presence of 0.1 .mu.M
ISO (l), 0.1 .mu.M PIA (P), 0.1 .mu.M DPCPX (D) or a combination of these
for 9 min as indicated. Each value represents the mean.+-.SE of 13
different membrane preparations. Asterisks denote a statistically
significant difference from the basal (B) value. The dagger denotes a
statistically significant difference from the ISO (l) value. The double
daggers denotes a statistically significant difference from the PIA+ISO
(PI) value.
FIG. 9(A and B) are graphs illustrating the effect of CGS-21680 on
ventricular myocyte (FIG. 9A) and myocardial (FIG. 9B) membrane adenylyl
cyclase activities. Membrane adenylyl cyclase activity was assessed in the
absence (.circle-solid.) or presence (.smallcircle.) of 0.1 .mu.M DPCPX or
DPCPX+10 .mu.M CGS-15943 (.DELTA.) at 0 to 1 mM CGS-21680, as indicated,
for 9 min. Each value represents the mean.+-.SE for 16 (FIG. 9A) and 14
(FIG. 9B) individual membrane preparations. Asterisks denote a
statistically significant difference from the appropriate zero CGS-21680
value. Daggers denote a statistically significant difference from the
corresponding value in the presence of DPCPX.
FIG. 10 is a graph illustrating the effect of N-ethylcarboxamidoadenosine
("NECA") on ventricular myocyte membrane adenylyl cyclase activity.
Cyclase activity was assessed in the absence (.circle-solid.) or presence
(.smallcircle.) of 0.1 .mu.M DPCPX or DPCPX+10 .mu.M CGS-15943 (.DELTA.)
at 0 to 1 mM NECA, as indicated, for 9 min. Each value represents the
mean.+-.SE for 11 individual membrane preparations. Asterisks denote a
statistically significant difference from the appropriate zero NECA value.
Daggers denote a statistically significant difference from the
corresponding NECA values in the absence of CGS-15943. Double daggers
denote a statistically significant difference from the corresponding NECA
values in the absence of DPCPX.
FIG. 11 is a graph illustrating the effect of naphthyl-substituted
aralkoxyadenosine ("SHA-082") on ventricular myocyte membrane adenylyl
cyclase activity. Cyclase activity was assessed in the absence
(.circle-solid.) or presence (.smallcircle.) of 0.1 .mu.M DPCPX at 0 to
0.1 mM SHA-082 for 9 min as indicated. Each value represents the
mean.+-.SE for 16 individual membrane preparations. The asterisk denotes a
statistically significant difference from the appropriate zero SHA-082
value.
FIG. 12 is a graph illustrating the effect of CGS-21680 on cAMP content of
cultured ventricular myocytes. Cultures were exposed for 10 min to 0 (B),
0.1 .mu.M (C7), 1 .mu.M (C6), or 10 .mu.M (C5) CGS-21680, 1 .mu.M
3,7-dimethyl-1-propargylxanthine ("DMPX") (D) or 1 .mu.M DMPX plus 10
.mu.M CGS-21680 (DC5). Myocyte cAMP levels were then determined as
described in the Methods. Each value represents the mean.+-.SE of 5
individual culture plates obtained from 16 separate preparations.
Asterisks denote a statistically significant difference from basal (B).
The dagger denotes a statistically significant difference from the
corresponding C5 and C6 value.
FIG. 13 is a graph illustrating the effect of 10.sup.-8 M isoproterenol (5
sec.) on LVP, +dP/dt.sub.max and -dP/dt.sub.max in the normoperfused
(control) and hypoperfused (hypoperf) mechanically depressed rat heart.
The values are the mean.+-.SE of six administrations performed in four
hearts. The hypoperfused values are statistically different from the
corresponding normoperfused values.
FIG. 14 is a depiction of a typical pressure recording illustrating the
effect of 2.times.10.sup.-5 M adenosine plus 2.times.10.sup.-7 M DPCPX on
LVP, +dP/dt.sub.max and -dP/dt.sub.max of the mechanically depressed (by
hypoperfusion) rat heart. The adenosine and DPCPX were administered for
1.5 minutes from the time indicated by the arrow.
FIG. 15 is a graph illustrating the effect of 2.times.10.sup.-5 M adenosine
plus 2.times.10.sup.-7 M DPCPX (1-2 min.) on LVP, dP/dt.sub.max and
-dP/dt.sub.max in the normoperfused (control) and hypoperfused (hypoperf)
mechanically depressed rat heart. The values are the mean.+-.SE of six
administrations performed in four hearts. The adenosine treatment values
in the hypoperfused hearts are statistically different from the
corresponding control values with adenosine.
DETAILED DESCRIPTION
This invention provides a simple, rapid and efficacious method for
increasing the contractile performance of a compromised myocardium. The
compromise may result from various factors, e.g., ischemia, hypoxia,
hypertrophy, old age, underlying heart disease of unknown cause, or
combinations thereof.
An adenosine receptor agonist, e.g., adenosine, can be used according to
this invention to treat a mammal that has been identified as having a
compromised myocardium. Mammals, and humans in particular, are known to
display various signs and symptoms relating to the existence of a
compromised myocardium. The recognition of such signs and symptoms is
within the skill of medical practitioners. Signs and symptoms of a
compromised myocardium in a human patient include, but are not limited to,
the following: decrease in ejection fraction, as determined by
echocardiography; increase in heart size, as determined by
echocardiography or X-ray; increase in diastolic pressure; dyspnea;
shortness of breath; and effusions around the heart, i.e., the
accumulation of fluid in the pericardial cavity.
This invention involves activating type A.sub.2 stimulatory adenosine
receptors, which have a low affinity for adenosine. Therefore, the
invention may be practiced with adenosine or any other compound that is an
A.sub.2 receptor agonist, i.e., that binds to the A.sub.2 receptor,
thereby causing signal transduction. The following compounds are examples
of adenosine A.sub.2 receptor agonists: adenosine,
carboxyethyl-phenethyl-amino-ethylcarboxamido-adenosine ("CGS-21680"),
N-ethyl-carboxamidoadenosine ("NECA"), naphthyl-substituted
aralkoxyadenosine ("SHA-082"), and 2-phenylaminoadenosine.
The following considerations may be useful in identifying additional
compounds that are adenosine A.sub.2 receptor agonists. The introduction
of an aromatic substituent at the amino group on the 6 position of the
adenine moiety increases adenosine A.sub.2 receptor selectivity (Bridges
et al., J. Med. Chem. 31:1281 (1988)). A C.sub.2 H.sub.5 --N--R
substituent at the 5' position plus a large aromatic substituent at the 2
position markedly increases selectivity for adenosine A.sub.2 receptors
(Hutchinson et al. J. Pharmacol. Exp. Thera. 251:47 (1989)). Also,
2-hexynyl 5'-ethyl-carboxamido additions enhance adenosine A.sub.2
receptor agonist activity (Monopoli et al., Arzneim.-Forsch Drug Res.
44:1296 (1994)).
Although type A.sub.2 receptors have a relatively low affinity for
adenosine, they may, but will not necessarily, have a low affinity for a
particular adenosine receptor agonist. An adenosine receptor agonist may
display specificity for type A.sub.2 receptors, or it may lack
specificity, activating type A.sub.1 receptors as well as type A.sub.2
receptors. If the invention is practiced with adenosine or a non-specific
adenosine receptor agonist, the level of adenosine or agonist in the
myocardium must be sufficiently elevated to activate the type A.sub.2
adenosine receptors. Insufficient elevation may activate type A.sub.1
adenosine receptors only, thereby decreasing contractile performance of
the heart.
In the practice of this invention, an adenosine A.sub.2 receptor agonist,
adenosine A.sub.1 receptor antagonist, adenosine transport inhibitor,
inhibitor of adenosine metabolism, or any combination thereof, may be
administered by any route suitable for delivery to the myocardium. The
preferred route of administration is intravenous. The adenosine or other
adenosine receptor agonist may be comprised in any pharmaceutically
acceptable composition. Preferably, intravenous adenosine is administered
as a pharmaceutically acceptable aqueous solution. Preferably, adenosine
is administered at a predetermined dose of between 1 and 140 .mu.g/kg/min.
More preferably, the predetermined adenosine dose is between 10 and 100
.mu.g/kg/min. Most preferably, the predetermined adenosine dose is between
20 and 40 .mu.g/kg/min. When the invention is practiced with adenosine, a
selected combination of dose level and route of administration preferably
yields a serum adenosine concentration between 10 nM and 10 mM. If
adenosine or other adenosine receptor agonist is administered in
conjunction with a second compound which potentiates the beneficial effect
of those compounds, the dose level of adenosine or adenosine or the
adenosine receptor agonist should be adjusted accordingly. It may be
necessary to begin with a predetermined dose of adenosine or adenosine
receptor agonist and then titrate the dosage according to one or more
selected clinical parameters, in order to achieve the desired increase in
contractile performance of the compromised myocardium. The dose level of
an adenosine receptor agonist will depend on various factors, including
the following: toxicity, in vivo half-life, affinity for the type A.sub.2
adenosine receptor and affinity for the type A.sub.1 adenosine receptor.
The administration of an adenosine A.sub.2 receptor agonist, adenosine
A.sub.1 receptor antagonist, adenosine transport inhibitor, inhibitor of
adenosine metabolism, or any combination thereof, can be singular or
intermittent, in response to acute signs or symptoms of myocardial
compromise. Alternatively, the administration can be continuous, for an
indefinite period, to sustain the performance of a chronically or
permanently compromised myocardium.
When adenosine or other adenosine A.sub.2 receptor agonist is administered
according to this invention, it is desirable to monitor one or more of the
compound's physiological effects. Such monitoring may be used, in a
particular individual, to verify increased myocardial contractile
performance, to identify undesirable side effects and, as noted above, to
adjust the dose level. Examples of potential undesirable side effects
associated with adenosine administration are flushing in the head and
neck, "uneasy" feelings, and transient cardiac arrest due to A-V nodal
blockade. Preferably the effects of adenosine or an adenosine receptor
agonist on a patient would be assessed by monitoring one or more of the
following: blood pressure, ease of patient's breathing, image of the heart
produced by echocardiography (ultrasound cardiography), and electrical
activity of the heart (electrocardiography).
Adenosine is normally present in micromolar amounts in the mammalian
myocardium, where it is involved in a variety of physiological and
biochemical processes. It is transported across biological membranes,
e.g., into or out of cells or organelles, by adenosine transport proteins
(also called "transporters" or "carriers"). One mechanism by which
interstitial adenosine is cleared (i.e., removed) is transport into cells,
where it is metabolized in various biochemical pathways. Therefore, high
levels of exogenous interstitial adenosine may be promoted initially or
prolonged, by administering, in conjunction with adenosine, a compound
that inhibits adenosine transport into cells. Accordingly, in a preferred
embodiment of this invention, an adenosine transport inhibitor is
administered in conjunction with adenosine administration. Examples of
adenosine transport inhibitors are dipyridamole,
S(4-nitrobenzyl)-6-thioinosine, S(4-nitrobenzyl)-6-thioguanosine and
Draflazine (see, Van Belle, Drug Devel. Res. 31:329, abstr. no. 1383
(1994). The adenosine transport inhibitor may be introduced into the
mammal by any suitable method, including via an oral, transmucosal,
intravenous, intramuscular or subcutaneous route. Alternatively, the
transport inhibitor may be inhaled by the mammal.
Another approach to inhibiting the clearance of interstitial adenosine is
the inhibition of adenosine metabolism. One pathway of adenosine
metabolism is the conversion of adenosine to inosine by adenosine
deaminase. Accordingly, in one embodiment of this invention, an adenosine
deaminase inhibitor is administered in conjunction with adenosine
administration. An example of an adenosine deaminase inhibitor is
erythro-9-(2-hydroxy-3-nonyl) adenine ("EHNA"). Another pathway of
adenosine metabolism is the conversion of adenosine to adenosine
monophosphate by adenosine kinase. Accordingly, in another embodiment of
this invention, an adenosine kinase inhibitor is administered in
conjunction with adenosine administration. An example of an adenosine
kinase inhibitor is iodotubercidin.
When adenosine is administered according to this invention, i.e, to
increase the contractile performance of a compromised myocardium via its
interaction with the type A.sub.2 receptors, the adenosine may be
simultaneously exerting a counterproductive effect via its interaction
with the type A.sub.1 receptors. Accordingly, in one embodiment of this
invention, a selective inhibitor of A.sub.1 receptor-mediated
(antiadrenergic) adenosine effects, i.e., an A.sub.1 receptor antagonist,
is administered in conjunction with adenosine administration. The
following compounds are examples of A.sub.1 receptor antagonists:
1-allyl-3,7-dimethyl-8-phenylxanthine, 8-cyclopentyl-1,3-dimethylxanthine,
8-cyclopentyl-1,3-dipropylxanthine ("DPCPX"),
1,3-diethyl-8-phenylxanthine, 8-phenyltheophylline and xanthine amine
congener ("XAC"). Similarly, an A.sub.1 receptor antagonist can be
administered in conjunction with an adenosine receptor agonist. This is
particularly desirable if the adenosine receptor agonist is not specific
for type A.sub.2 adenosine receptors.
Chemical compounds useful in practicing this invention or used in the
experiments described below are often known in the art primarily by
abbreviations, common names or arbitrary designations, several of which
are collected in Table 1.
TABLE 1
______________________________________
NOMENCLATURE AND ACTIVITY OF
SELECTED AGONISTS AND ANTAGONISTS
ABBREVIATION/
CHEMICAL RECEPTOR/
COMMON NAME COMPOUND ACTIVITY
______________________________________
CGS-21680 2-p-(2-carboxy-ethyl)
A.sub.2 agonist
phenethyl-amino-5'-N-
ethylcarboxamido-
adenosine
CGS-15943 9-chloro-2-(2-furanyl)-5,6-
A.sub.2 antagonist
dihydro-1,2,4-triazolo-(1,5-
C)quinazolin-5-imine
CSC 8-(3-chlorostyryl)caffeine
A.sub.2 antagonist
DMPX 3,7-dimethyl-1-(2-
A.sub.2 antagonist
propargyl)xanthine
DPCPX 1,3-dipropyl-8- A.sub.2 antagonist
cyclopentylxanthine
NECA N-ethylcarboxamido-
A.sub.2 agonist
adenosine
ISO Isoproterenol .beta.-adrenergic
agonist
SHA-082 naphthyl-substituted
A.sub.2 agonist
aralkoxyadenosine
PIA phenylisopropyladenosine
A.sub.1 agonist
______________________________________
EXPERIMETNAL INFORMATION
Ventricular Myocyte Study
Primary cultures of adult rat ventricular myocytes were prepared
essentially according to previously described methods (Romano et al., Am.
J. Physiol. 257:H1088 (1989)). Two male Sprague-Dawley rats (Charles
River, Wilmington, Mass. or Harlan, Indianapolis, Ind.) were decapitated,
and the hearts were rapidly excised and constant pressure (70 cm H.sub.2)
and nonrecirculated) perfused for 10 minutes through the aortas with
filtered (0.45 .mu.m membrane filter) perfusing solution (NaCl, 118 mM;
glucose, 10 mM; NaHCO.sub.3 25 mM; KCl, 4.69 mM; MgSO.sub.4 1.18 mM; and
KH.sub.2 PO.sub.4, 1.18 mM; (pH 7.4)), to which 2.5 Mm CaCl.sub.12 was
added, at 37.degree. C. After equilibration, the hearts were constant
pressure perfused with fresh physiological saline ("PS") containing no
added Ca.sup.2+ until spontaneous contractions ceased (i.e., .about.30
seconds). The hearts were then perfused for 4-10 minutes in a
nonrecirculating manner with PS containing 0.73 mg/ml collagenase, 0.16
mg/ml hyaluronidase, 1 mg/ml recrystallized bovine serum albumin ("BSA")
and 48.4 .mu.M Ca.sup.2+ at a rate of 3-4 ml/min/heart. Ventricles, free
of atria, were removed from the perfusion system, cut into 8 pieces and
placed in a 50 ml Erlenmeyer flask with 5 ml of PS containing 0.73 mg/ml
collagenase, 0.16 mg/ml hyaluronidase, 2.5 mg/ml BSA, and 50 .mu.M
Ca.sup.2+ (incubation solution). The flask was gently shaken (40
cycles/min) in a reciprocating water bath with continuous gassing (95%
O.sub.2, 5% CO.sub.2) for 7 minutes at 37.degree. C. The incubation
solution was aspirated and this shaking procedure was repeated with fresh
incubation solution 3-5 times depending on the likelihood that the tissue
was ready to dissociate. After the final incubation period, the solution
was aspirated and replaced with 10 ml of fresh incubation solution. The
flask was then shaken rapidly (120 cycles/min) for 10 minutes with gassing
to dissociate the myocytes. The flask contents were filtered through a 250
.mu.m nylon mesh into a 50 ml polypropylene centrifuge tube, to which 40
ml of PS containing 5.95 mg/ml BSA and 99.7 .mu.m Ca.sup.2+ (wash
solution) was gradually added.
The myocytes were allowed to settle for 15 minutes, and the upper two
thirds of the wash solution was aspirated. Upon the addition of 30-35 ml
of wash solution, this settling step was repeated. The wash solution was
aspirated and the myocyte pellet was resuspended in 10 ml of minimum
essential medium ("MEM"). The myocyte suspension was allowed to settle for
5 minutes and then brought to a final volume of 22 ml with MEM. Two
milliliters of the myocyte suspension was seeded onto each of 60 mm
culture dishes containing 2 ml of MEM. Because myocytes do not attach
under these plating conditions, these myocytes were used for contractile
experiments.
When myocytes were to be used for biochemical studies, each of the culture
dishes was preincubated for 2 hours with 1 ml of MEM containing 33 .mu.g
of laminin in a 37.degree. C. incubator gassed with 5% CO.sub.2 in room
air. The laminin solution was removed prior to myocyte seeding. After
seeding, the dishes were incubated for an additional 2 hours. The settling
and short term culture procedures were performed to purify the myocytes so
that >95% of the myocytes adhering to the dishes were rod shaped.
The contractile function of individual myocytes was assessed by dispersing
50-100 cells in a 506 .mu.l myocyte chamber (11.times.23.times.2 mm deep).
The chamber (FIG. 1) was continuously suffused (850 .mu.l/min) with fresh
suffusion solution ("SS") in mM: 136.4 NaCl, 4.7 KCl, 1.0 CaCl.sub.2, 10
hydroxyethylpiperzine-ethanesulfonicacid (HEPES), 1.0 NaHCO.sub.3, 1.2
MgSO.sub.4, 1.2 KH.sub.2 PO.sub.4, 10 glucose, 0.6 ascorbate, 1.0
pyruvate) at 20.degree. C. The chamber was mounted on an inverted
microscope stage and contained platinum wire electrodes for initiating
myocyte contraction at 0.5 Hz (voltage 10% above threshold for 5 msec
duration).
The image of the myocyte was projected via an inverted microscope at
300.times. onto a line scan camera (Fairchild, Model 1600R) containing a
linear array (1.times.3456) of photodiodes. When aligned with the
longitudinal axis of the cell the camera detected the movement of the 2
ends of the myocyte upon contraction (shortening and relengthening). The
signals from the line scan camera were displayed on an oscilloscope
(Hitachi, Model V-660) which permitted optimal positioning of the camera
over the myocyte. When the myocyte was transilluminated, both ends of the
cell were easily discriminated. Myocyte length and length change with
respect to time for a single contraction was measured by determining the
pixels in which the appropriate transitions between light and dark
occurred. The line scan camera was calibrated with a stage micrometer
scaled in 10 .mu.m divisions. The signals from the camera were directed to
a Hewlett Packard computer (Model Vectra RS/20C). Custom computer
programming (MCS Computer Consulting, Keene N.H.) permitted determination
of myocyte length (L), maximum length change (shortening) with contraction
("LC"), duration of shortening (DS), time-to-peak shortening ("TPS"), time
to 75% relaxation (relengthening, "TR") and the rate of maximal shortening
(+dL/dt.sub.max) and relaxation (-dL/dt.sub.max). An example of edge
movement detection for a single contraction of a myocyte is shown in FIG.
2A. Also illustrated as a function of time is L, .DELTA.L and + and -dL/dt
in FIGS. 2B, 2C, and 2D, respectively.
After a 3-4 hour incubation period, myocytes were harvested by placing the
culture dishes on ice; aspirating and discarding the culture medium;
scraping the attached cells into 1 ml of ice cold buffer (pH 7.4)
containing 10 mM HEPES and 1 mM dithiothreitol (DTT); and transferring the
mixture to a 40 ml centrifuge tube. The culture dishes were rinsed twice
with buffer, and the rinses were added to the centrifuge tube. The
myocytes were centrifuged at 45,000 x g for 45 minutes and the supernatant
was discarded. The pellet was resuspended in 40 mM HEPES (pH 7.4) to yield
3.5-4 mg protein/ml and homogenized with a small clearance Dounce tissue
grinder (8 strokes). The membranes were centrifugated as described above,
resuspended (3-4 mg protein/ml) in 40 mM HEPES (pH 7.4), and assayed for
adenylyl cyclase activity immediately or stored at -80.degree. C. and
assayed within 10 days.
Crude ventricular myocardial membranes were prepared according to the
following procedure. Isolated rat hearts were perfused with 5 ml of ice
cold saline (0.9% NaCl) to wash out the blood, minced into 2-3 mm.sup.3
cubes, and placed in 10 ml of homogenization buffer (HB) containing 10 mM
HEPES (pH 7.4), 1 mM ethylenediamine-tetraacetic acid (EDTA), 1 mM DTT and
10 .mu.g/ml soybean trypsin inhibitor. The suspension was homogenized with
a PT-10 Polytron.TM. generator at a speed of 6 for two 15-second periods
separated by 15 seconds. The homogenized material was also treated with 2
strokes of a glass/teflon motor driven Potter-Elvehjen homogenizer
operated at 1/2 full speed. Upon addition of 4.7 ml of HB containing 1.25M
sucrose the homogenate was mixed and centrifugated at 1,000 x g for 15
minutes. The supernatant was filtered through 4 layers of cheesecloth and
14.5 ml HB was added. The mixture was centrifugated at 45,000 x g for 45
minutes and the pellet was suspended in 3-5 ml of 40 mM HEPES buffer (pH
7.4) with the small clearance Dounce tissue grinder (6 strokes) to yield
3-5 mg protein/ml. Membranes were assayed immediately for adenylyl cyclase
activity. All preparative steps for the crude membranes were performed at
0.degree.-1.degree. C.
Protein concentration was determined by a standard method (BCA Protein
Assay, Pierce, Rockford, Ill.) using BSA as a standard. Each 60 mm culture
dish contained 200-600 .mu.g of adhering rod shaped myocyte protein
resulting in a total of 3-6 mg of protein from a pair of hearts.
The assay system used to measure adenylyl cyclase activity minimizes the
formation of endogenous adenosine. The assay used was essentially as
described by Romano et al. (supra). Myocyte membranes (15-25 .mu.g
protein) were incubated in 50 .mu.l of a buffer containing 40 mM HEPES (pH
7.4), 5 mM MgCl.sub.2, 1 mM DTT, 5.5 mM KCl, 0.1 mM 2'-deoxy-cAMP (dcAMP),
0.1 mM 2'-deoxy ATP (dATP), 20 mM phosphoenolpyruvate, 2 units pyruvate
kinase, 0.25 units adenosine deaminase, 1 mM ascorbic acid, 100 mM NaCl,
0.1 mM ethyleneglycol-bis (.beta.-aminoethylether)
N,N,N',N',-tetroaceticacid (EGTA), 10 .mu.M guanosine 5'-triphosphate
(GTP), and .about.2.times.10.sup.6 counts/minutes of [.alpha.-.sup.32
P]dATP for 20 minutes at 30.degree. C. The reaction was stopped by adding
40 .mu.l of a solution containing 2% sodium dodecyl sulfate (SDS), 45 mM
ATP, 1.3 mM cAMP, and [.sup.3 H]dcAMP (.about.4000 count/min) and by
boiling for 2 minutes. The formed [.alpha.-.sup.32 P]dcAMP was separated
from the [.alpha.-.sup.32 P]dATP by sequential chromatography using
columns of cation exchange resin AG 50W-X4 (200-400 mesh) and neutral
alumnia AG 7 (100-200 mesh) after the methods of Salomon (Adv. Cyclic
Nucleotide Res. 10:35 (1979). All results were corrected for column
recovery of [.sup.3 H]dcAMP, which ranged between 60 and 90%. The activity
of the adenylyl cyclase is expressed as pmol [.alpha.-.sup.32 P]dcAMP
formed/min/mg protein.
Cyclic AMP levels were determined in cultures of ventricular myocytes
according to the following method. After the 2-hour incubation period, the
MEM was aspirated and replaced with 2 ml of fresh MEM. Adenosine agonists,
ISO, or adenosine antagonists were added to the medium bathing the cells
at the concentrations and times indicated. The experiment was terminated
by removing the medium from the dish and adding 200 .mu.l of 1N HCl over
the cultured myocyte surface. The dishes were then frozen in liquid
N.sub.2 and stored at -70.degree. C. or held on ice momentarily until
extraction was initiated.
For assay, the contents of the dishes were transferred (using 1 ml of
distilled/deionized H.sub.2 O) into microcentrifuge tubes. The extracts
were heated for 1 hour at 57.degree. C. and sonicated for 10 minutes. The
extracts were then centrifugated at 14,000 x g for 15 minutes. The
supernatant was removed, evaporated, reconstituted in 500 .mu.l of 50 mM
sodium acetate buffer and assayed for cAMP using an .sup.125 I-cAMP RIA
kit (Amersham). The pellet was solubilized with 0.1N NaOH and protein
determined. The cAMP values are reported in pmol cAMP/mg protein of the
extract pellet (total cell protein). This cAMP assay procedure routinely
provided recovery values in excess of 90%.
All data are expressed as means.+-.one standard error of the mean (SE). The
concentration of agonist that produced 50% of the maximum stimulatory
response (EC.sub.50) was determined from nonlinear regression analysis.
Statistical analysis was performed on actual (not normalized) data.
Statistical significance was determined using one-way independent analysis
of variance. A probability (P value) of less than 0.05 was accepted as a
statistically significant difference.
Ventricular Myocyte Results
An adenosine A.sub.1 receptor agonist, phenylisopropyladenosine ("PIA"),
reduced .beta.-adrenoceptor-mediated increases in ventricular myocyte
contractility, caused by isoproterenol. The A.sub.1 receptor-mediated
inhibition of .beta.-adrenoceptor-mediated increases in contractility was
reduced by an adenosine A.sub.1 receptor antagonist. In individual
contracting rat ventricular myocytes ISO at 0.2 .mu.M increased by 61, 63
and 100% the maximum length change (shortening) with contraction (LC) and
maximum rates of shortening (+dL/dt.sub.max) and relaxation
(-dL/dt.sub.max), respectively (Table 2). These increases were accompanied
by decreases of 30, 14 and 39% in the duration of shortening ("DS"),
time-to-peak shortening ("TPS") and time to 75% relaxation ("TR"),
respectively. A typical recording depicting the length changes associated
with individual myocyte contractions under these conditions is given in
FIG. 3. An adenosine A.sub.1 receptor agonist, PIA, at 2 .mu.M reduced the
ISO-induced increases or decreases in LC, DS, TPS, TR, +dL/dt.sub.max and
-dL/dt.sub.max by 91, 47, 82, 37, 60 and 78%, respectively. An adenosine
A.sub.1 receptor antagonist, DPCPX, at 0.2 .mu.M reversed the PIA
reduction of the ISO induced contractile responses.
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