Department of Pharmacology (M.F., P.D.), University of Padova,
Padova, Italy;
Institute of Pharmacology (P.A.B., S.G.), University of
Ferrara, Ferrara, Italy;
Institute of Pharmaceutical Sciences (L.M.,
P.F.), University of Genova, Genova, Italy
In electrically driven left atria isolated from guinea pig and rat, a
new milrinone analog, 6-ethyl-5-propionyl-1,2-dihydro-2-oxo-3-pyridine carbonitrile, produced a positive inotropic effect that was not dependent on adrenergic mechanisms and was more marked than that exerted by the parent compound. Its inotropic action was almost completely abolished by pretreatment of atria with adenosine deaminase and correlated well with its binding ability to the cardiac adenosine A1 receptor. In this regard, the analog showed a 100-fold
higher affinity for adenosine receptor than that of milrinone.
Moreover, it shifted to the right the concentration-response curves for the negative inotropic action of the stable adenosine receptor agonist
R-phenylisopropyladenosine. The new analog behaved as a competitive
inhibitor of Type III phosphodiesterase isolated from both guinea pig
and rat, although its Ki value was 10 times higher than that of milrinone. However, an increase in cAMP levels does
not seem to be involved in the mechanism of action of the new compound,
because the presence of carbachol did not decrease the extent of the
positive inotropic effect of the analog and did not modify its
EC50 in either guinea pig or rat myocardial preparations.
Taken together, these results suggest that the milrinone structure can
be modified, giving rise to a more active compound whose inotropic
effect in both guinea pig and rat appears to be more clearly related to
antagonism toward endogenous adenosine than to Type III
phosphodiesterase inhibition.
 |
Introduction |
The
treatment of congestive heart failure is largely based on the use of
cardiac glycosides, diuretics and vasodilators. Although digoxin is one
of the most commonly prescribed drugs, the role of digitalis in the
management of the disease remains at the center of the oldest
continuing controversy in the history of medicine (Packer, 1997
). The
pronounced toxic effects and low therapeutic index of digitalis
glycosides (Mason et al., 1971; Lathers and Roberts, 1980)
have stimulated the search for a drug with positive inotropic effects
but minimal side effects (e.g., influence on cardiac
rhythm). A new class of cardiotonics, of which the parent compound is
amrinone, has been proposed as a replacement for digitalis (Farah and
Alousi, 1978; Alousi et al., 1979; Ward et al.,
1983). These drugs have both inotropic and peripheral vasodilator
activities and therefore enhance cardiac output by increasing cardiac
contractility and simultaneously reducing impedence to ventricular
ejection (Miller et al., 1981; Taylor et al.,
1982). Amrinone and its analog milrinone are referred to as inhibitors
of the low-Km cGMP-inhibited cAMP PDE, also
called Type III PDE (Nicholson et al., 1991), and their
positive inotropic and chronotropic effects are ascribed to an increase
in cAMP levels (Honerjaeger et al., 1981; Endoh et
al., 1982; Earl et al., 1986). However, because close
correlation between inhibition of Type III PDE and inotropic effect is
lacking in various animal species (Alousi and Farah, 1980; Carpenedo
et al., 1984; Kobylarz et al., 1985), other
biochemical mechanisms have been proposed to explain the cardiac action
of amrinone and milrinone (Alousi et al., 1979; Azari and
Huxtable, 1980; Kenakin and Scott, 1987; Parson et al.,
1988). In particular, antagonism toward endogenous adenosine at the
cardiac A1 inhibitory receptor has been suggested (Dorigo
and Maragno, 1986; Earl et al., 1986; Dorigo et
al., 1990, 1992). Inhibition of the binding of adenosine to
A1 receptor may produce a positive inotropic effect without marked variations in cAMP levels and without the related risk of
arrhythmias (Lubbe et al., 1992). The possibility of
antagonism toward endogenous adenosine is also particularly important
because adenosine, released in large amounts during heart failure
(Newman et al., 1984), may further damage the heart by
slowing conduction in the sinoatrial and AV nodes and reducing atrial
contractility. For these reasons, the search continues for new
adenosine antagonists more active than amrinone and milrinone.
In our previous studies (Dorigo et al., 1991, 1992, 1993,
1996), we used milrinone as the parent compound, variously modified its
molecule and obtained several compounds that enhanced cardiac contractility to various extents. A linear relationship was observed between the ability of these new compounds to displace adenosine from
its receptor and their inotropic action (Dorigo et al.,
1992, 1997). Taking into account some observations of a
structure-activity relationship (Dorigo et al., 1997), we
designed and synthesized a new milrinone analog,
6-ethyl-5-propionyl-1,2-dihydro-2-oxo-3-pyridine carbonitrile, that is
closely related to the parent compound. In the present study, we
investigated and characterized the cardiac effects of this new molecule
on isolated guinea pig and rat atria. In order to understand the
biochemical mechanisms responsible for the cardiac effects of the new
milrinone analog, we also determined the binding of the compound to
cardiac A1 receptors and its inhibitory effect on Type III
PDE isolated from guinea pig and rat ventricular tissue. All studies
were carried out on tissues isolated from both guinea pig and rat,
because Azari and Huxtable (1980) observed a species difference in the
action of the parent drug amrinone on Langendorff perfused heart
isolated from guinea pig and rat.
| |
Materials and Methods |
Myocardial preparations.
Normal or reserpine-treated (2 mg/kg i.p. daily for 2 days) guinea pigs (300-500 g b.wt.) and rats
(150-200 g b.wt.) were killed by a blow to the head followed by
exsanguination. The atria were separated from the ventricles and
suspended vertically in a 30-ml organ bath containing a physiological
salt solution constantly gassed by 95% O2 and 5%
CO2 at 29°C. The bath solution contained (mM): NaCl 120, KCl 2.7, CaCl2 1.36, MgCl2 0.09, NaH2PO4 0.4, NaHCO3 12 and glucose
5.5.
The atria were electrically driven at 1 Hz by square-wave pulses just
above threshold voltage, 0.6 to 0.9 msec in duration (S44 stimulator,
Grass Instrument Corporation, Quincy, MA). The force of contraction was
recorded on an isometric force transducer (7003 Basile, Comerio,
Varese, Italy) connected to a rectilinear recorder (KV 135 Battaglia
Rangoni, Casalecchio di Reno, Bologna, Italy). The initial
equilibration period was 40 to 60 min for each preparation. Resting
tension was adjusted to about 5 mN.
In myocardial preparations isolated from reserpine-treated animals,
depletion of catecholamines was confirmed by the lack of any positive
inotropic effect of tyramine (15 µM). Where indicated, propranolol (1 µM), carbachol (50 nM) or ADA (2 U/ml) was added to the bath medium
20 min before addition of the drugs.
In each atrial preparation, the response to isoprenaline (0.2 and 0.02 µM for guinea pig and rat atria, respectively) was determined before
addition of the test compounds and was considered the maximum positive
inotropic effect (Emax) obtainable in the atria.
Milrinone analog was added cumulatively, and the inotropic response
caused by each drug concentration was recorded up to the maximum
response before a higher concentration was added. The peak of the
inotropic response caused by each concentration of analog was reached
within 10 min. The effect of the drug was defined as the difference
between the force of contraction before and after its addition to the
bathing fluid and was expressed as a percentage of the maximum response
(Emax) induced by isoprenaline in the same
preparation. The EC50 value was graphically determined and
was the concentration that gave half the maximum effect obtainable with
the drug.
Milrinone and its analog were dissolved in DMSO, the final
concentration of which in the medium did not itself influence the basal
activity of the atrial preparations.
Receptor binding assay.
Guinea pig and rat ventricular
tissue was dissected in a Brinkman PT-10 Polytron (setting 6) in 25 volumes (w/v) of 50 mM Tris-HCl (pH 7.4). The homogenate was
centrifuged at 40000 × g and resuspended in 50 mM
Tris-HCl (pH 7.4) containing 2 U/ml ADA. After 30 min of incubation at
37°C, in order to metabolize endogenous adenosine, the membranes were
centrifuged at 40000 × g and the pellets were stored
at 
80°C until used.
Binding of milrinone analog to cardiac A1 adenosine
receptor was determined by its ability to displace
[3H]DPCPX, a specific antagonist for A1
receptors. Binding experiments were carried out for 150 min at 0°C in
1 ml of buffer containing 1 nM [3H]DPCPX, membranes from
10 mg (wet weight) of tissue and increasing concentrations of milrinone
analog. To determine IC50 values (where IC50 is
the concentration that displaces 50% of the labeled ligand), six
different concentrations (from 32 nM to 32 µM) of milrinone analog
were added to the binding assay medium. All experiments were carried
out in triplicate. Ki values
(Ki = inhibitory binding constant) were
calculated from the Cheng and Prusoff (1973) equation with
Ki = IC50/(1 + C*/Kd*), where C* is the
radioligand concentration used and Kd* (1 nM) is
its dissociation constant. Binding data were analyzed using the
nonlinear regression curve-fitting computer program Ligand (Munson and
Rodbard, 1980). Nonspecific binding was determined in the presence of
10 µM N6-cycloexyladenosine (or 1 mM theophilline) and
was routinely 30% of total binding. Bound and free
[3H]DPCPX were separated using a Brandel cell harvester
by rapid filtration under vacuum through Whatman GF/B glass-fiber
filters and then were washed three times with ice-cold buffer, dried
and counted in 5 ml of Istagel (Packard, Groningen, The Netherlands) in
a Beckmann Liquid Scintillation Spectrometer, at a counting efficiency
of about 55%.
Assay of soluble Type III PDE activity from guinea pig and rat
heart.
Type III PDE was isolated from guinea pig and rat heart
using the procedure described by Weishaar et al. (1986).
Type III PDE from guinea pig and rat heart had apparent
Km values for cAMP of 1.33 ± 0.15 and
1.37 ± 0.35 µM and Vmax values of
4.54 ± 0.29 and 4.20 ± 0.12 nmol/mg protein/min,
respectively. When assayed at 0.4 µM cAMP, the activities of guinea
pig and rat cardiac Type III PDE were inhibited by about 80% by 4 µM
cGMP. Both fractions were insensitive to calmodulin and were only
slightly inhibited by 100 µM rolipram, the specific inhibitor of Type
IV PDE.
PDE activity was measured by the two-step procedure of Thompson
et al. (1974), as previously described (Dorigo et
al., 1992).
Assay of ATP-dependent 45Ca++ uptake by
cardiac sarcoplasmic reticulum vesicles.
A crude cardiac membrane
vesicle preparation enriched in sarcoplasmic reticulum was obtained by
the method of Jones et al. (1977) from guinea pig and rat
ventricular tissue. Ca++ uptake was determined as
previously described (Floreani et al., 1996). Milrinone and
milrinone analog were dissolved in DMSO; the same amount of DMSO was
always added to the controls.
Assay of Na+/K+ ATPase, Ca++
ATPase and Na+/Ca++ exchange carrier activities
in cardiac sarcolemmal vesicles.
Cardiac sarcolemmal vesicles were
prepared from guinea pig and rat ventricular tissue by the method of
Slaughter et al. (1983). Na+/K+
ATPase, Ca++ ATPase and Na+/Ca++
exchange carrier activities were measured as previously described (Floreani et al., 1996).
Protein assay.
Protein content was determined according to
Lowry et al. (1951) using bovine serum albumin as standard.
Statistical analysis.
Data are expressed as arithmetic
means ± S.E.; Student's two-tailed t test was used
for statistical analysis.
Chemicals.
The following drugs and chemicals were used in
this study: milrinone (Sanofi-Winthrop, Collegeville, PA), milrinone
analog (L. Mosti, Institute of Pharmaceutical Sciences, University of Genova, Italy), reserpine, tyramine, propranolol, isoprenaline, R-PIA,
carbamylcholine chloride (carbachol), ADA (type VI, from calf
intestinal mucosa), Tris, ATP, cAMP, cGMP, EGTA, DMSO,
N6-cycloexyladenosine, Dowex 1 × 2, DEAE cellulose
and 5
-nucleotidase (grade II, from Crotalus atrox) (Sigma
Chemical Co., St. Louis, MO), [3H]DPCPX (NEN New England
Nuclear, Florence, Italy) and 8-[3H] cAMP (Amersham
Italia, Milan, Italy). All other reagents were of analytical grade.
| |
Results |
The newly synthesized analog of milrinone was closely related to
the parent drug. As shown in figure 1,
the basic structure of dihydro-oxo-pyridine carbonitrile was
maintained, whereas the 4-pyridinyl moiety in C5 and the methyl group
in C6 were substituted with a propionyl group and an ethyl group,
respectively.
Effect of milrinone analog on atria contractility.
We
determined the effect of the new analog on the contractility of
isolated guinea pig and rat atria (n = 8) electrically driven at 1 Hz. It caused a concentration-dependent increase in the
force of contraction of atria from both species. The increase was quite
rapid in onset and reached its peak within 10 min. Figure 2 shows the cumulative
concentration-response curves for the positive inotropic effect of
milrinone analog in guinea pig (panel A) and rat (panel B) electrically
driven left atria, compared with the effect of the parent drug. When
the extent of contraction induced by the drugs was referred to the
maximum contraction induced by isoprenaline in the same experimental
conditions in both guinea pig and rat atria, the highest concentrations
of the analog appeared to be more active than those of the parent
compound. Moreover, the analog caused a more marked increase in the
force of contraction of rat atria than of guinea pig atria. The
EC50 values for milrinone and its analog, calculated from
the cumulative concentration-response curves, indicate that the potency
of the analog was quite similar to that of the parent compound in both
species. The effect of the new compound was not modified in
catecholamine-depleted atria or in atria pretreated with 1 µM
propranolol (data not shown).
View larger version (16K):
[in this window]
[in a new window]
|
Fig. 2.
Cumulative concentration-response curves for the
positive inotropic effect of milrinone ( ) and milrinone analog ( )
on isolated guinea pig (panel A) and rat (panel B) atria, electrically
driven at 1 Hz at 29°C. Effects, measured at time of maximum effect, were calculated as differences in force of contraction before and after
drug addition and were expressed as percentages of maximum increase in
force of contraction (Emax) induced by
isoprenaline in the same preparation. Predrug force of contraction:
5.3 ± 0.4 mN and 4.1 ± 0.65 mN in guinea pig and rat,
respectively. Isoprenaline-induced increase in force of contraction:
9.8 ± 1.3 mN (+185% vs. control) and 8.5 ± 1.1 mN (+207% vs. control) in guinea pig and rat, respectively. Values are mean ± S.E. from eight experiments carried out on
different myocardial preparations (n = 8).
|
|
Even at the highest concentration tested (1 mM), the analog did not
have toxic effects on the myocardial preparations; it did not cause
arrhythmias or any increase in resting tension. Moreover, the effect of
the analog was completely reversible; washout of myocardial
preparations restored the pre-drug force of contraction of atria.
When tested on spontaneously beating guinea pig and rat atria, the new
compound exerted a positive inotropic action quantitatively similar to
that observed in electrically driven left atria (data not shown).
Because antagonism toward endogenous adenosine has been suggested as
one of the mechanisms responsible for the positive inotropic effect of
some milrinone analogs (Dorigo et al., 1992, 1997), some
experiments were performed in the presence of ADA, the enzyme that
inactivates endogenous adenosine by converting it to inosine. The
addition of the enzyme (2 U/ml) to left atria isolated from both guinea
pig and rat evoked by itself a sustained increase in force of
contraction that lasted for 15 to 20 min and left the heart preparation
stabilized at a higher contractile level than in controls (+15% and
+30% in guinea pig and rat, respectively). Pretreatment of myocardial
preparations with ADA markedly decreased the effect of the milrinone
analog (table 1). As previously reported (Dorigo et al., 1990), in the same experimental conditions,
the effect of milrinone was also significantly diminished by depletion of endogenous adenosine. Control experiments confirmed that the treatment of atria with ADA did not alter the response of myocardial preparations to another agonist, isoprenaline (Dorigo et
al., 1990).
View this table:
[in this window]
[in a new window]
|
TABLE 1
Effect of ADA on the positive inotropic effect caused by milrinone
analog in guinea pig and rat electrically driven left atria
ADA (2 U/ml) was added to the bathing medium of electrically driven
left atria 20 min before the addition of the milrinone analog. Because
ADA itself increased the force of contraction of the atria, the effect
of the analog was evaluated by considering that the basal level of
force of contraction was that reached when the effect of ADA had become
stable. The effects, measured at the time of maximum effect, were
expressed as percentages of maximum increase in force of contraction
(Emax) induced by isoprenaline in the same
preparation. The values are means ± S.E. from four experiments
carried out on different myocardial preparations (n = 4).
|
|
To elucidate the involvement of adenosine antagonism further, we
evaluated the influence of the new compound on the negative inotropic
effect induced in atria by R-PIA, a stable adenosine receptor agonist.
As shown in figure 3, the analog caused a
rightward shift of the concentration-response curve for R-PIA. This
effect was more pronounced in rat (panel B) than in guinea pig (panel A).
View larger version (14K):
[in this window]
[in a new window]
|
Fig. 3.
Cumulative concentration-response curves for the
negative inotropic effect of R-PIA in guinea pig (panel A) and rat
(panel B) left atria in the absence () and presence () of 20 µM
analog. In experiments carried out with analog, myocardial preparations were treated for 10 min with 20 µM analog before the addition of
increasing concentrations of R-PIA. Values are means ± S.E. from
four experiments carried out on different myocardial preparations (n = 4).
|
|
To evaluate the involvement of cAMP increase in the mechanism of the
cardiotonic action of the new analog, we tested the influence of
carbachol (50 nM) on the positive inotropic effect caused by the
compound. The results indicated that the presence of carbachol did not
modify the EC50 values for the positive inotropic effect of
the analog in either guinea pig (EC50 = 40 ± 3 µM)
or rat (EC50 = 61 ± 5 µM) heart. Moreover, the
Emax of the analog was slightly enhanced, rather
than decreased, by pretreatment of myocardial preparations with
carbachol. This enhancement of Emax was probably related to the decrease in basal contractility observed in the presence
of carbachol.
Binding of milrinone analog to cardiac A1
receptors.
To confirm the involvement of antagonism toward
endogenous adenosine in the positive inotropic action of the new
milrinone analog, we determined the effect of the compound on the
specific binding of an adenosine A1 receptor antagonist,
[3H]DPCPX, in membranes from guinea pig and rat cardiac
tissue. Figure 4 clearly shows that the
analog displaced [3H]DPCPX from its binding sites to
isolated cardiac membranes. Using the Cheng and Prusoff (1973)
equation, we obtained Ki values of 1.30 ± 0.28 and 0.66 ± 0.03 µM in guinea pig and rat cardiac membranes, respectively.
View larger version (13K):
[in this window]
[in a new window]
|
Fig. 4.
Displacement curves for [3H]DPCPX bound
to guinea pig ( ) and rat ( ) cardiac membranes by increasing
concentrations of milrinone analog. Binding experiments were performed
for 150 min at 0°C in the presence of 1 nM [3H]DPCPX.
Experimental conditions are described in "Materials and Methods."
Values are means ± S.E. from three experiments carried out in
duplicate.
|
|
Effect of milrinone analog on Type III PDE activity.
Because
cardiac Type III PDE has been proposed as the target of bipyridine
cardiotonic drugs such as amrinone and milrinone, we tested the analog
on Type III PDE isolated from guinea pig and rat cardiac tissue. It
inhibited, in a concentration-dependent way, the activity of the enzyme
isolated from the cardiac tissue of both animal species.
Ki values of 11 µM and 19 µM were calculated for guinea pig and rat, respectively, as shown by the Dixon plots (Dixon, 1953) reported in figure 5.
Analysis of the data according to Lineweaver and Burk (1934) (fig.
6) clearly indicates that the analog
behaved as a competitive inhibitor for Type III PDE activity.
View larger version (11K):
[in this window]
[in a new window]
|
Fig. 5.
Dixon plots for the inhibitory activity of milrinone
analog on Type III PDE activity partially purified from guinea pig
(panel A) and rat (panel B) cardiac tissue. Enzyme activity was
evaluated in the presence of 0.4 µM () and 1 µM () cAMP. On
the ordinate, values are expressed as nmol/mg protein/min. Values are
means ± S.E. from three experiments carried out in duplicate on
different enzyme preparations (n = 3).
|
|
View larger version (8K):
[in this window]
[in a new window]
|
Fig. 6.
Lineweaver-Burk plots for the inhibitory activity of
20 µM milrinone analog () on Type III PDE activity partially
purified from guinea pig (panel A) and rat (panel B) cardiac tissue.
Enzyme activity was evaluated in the presence of increasing
concentrations (from 0.1 to 1 µM) of cAMP. On the ordinate, values
are expressed as nmol/mg protein/min. Results are means ± S.E.
from three experiments carried out in duplicate on different enzyme
preparations (n = 3).
|
|
Effect of milrinone analog on other enzymes and transport systems
involved in cardiac contractility.
The milrinone analog was also
tested on other enzymes and transport systems involved in the control
of cardiac contractility, such as the sarcolemmal
Na+/K+ ATPase, Ca++ ATPase and
Na+/Ca++ exchange carrier activities and the
sarcoplasmic reticulum Ca++ pump. It had no effect on them
(data not shown).
| |
Discussion |
The present results show that a new milrinone analog,
6-ethyl-5-propionyl-1,2-dihydro-2-oxo-3-pyridine carbonitrile, closely related to the parent compound, increases in a concentration-dependent way the force of contraction of isolated guinea pig and rat atria. The
new compound is more active than the parent drug in both types of
atria, although its potency is quite similar to that of milrinone. Its
cardiac action is completely independent of direct or indirect adrenergic mechanisms. In fact, it is not modified by pretreatment of
myocardial preparations with the beta blocker propranolol or by depletion of cardiac adrenergic stores by reserpine. Our data suggest that the main mechanism responsible for the positive inotropic effect of this milrinone analog is antagonism toward endogenous adenosine.
Several observations support this hypothesis. First, the analog binds
with high affinity to the A1 adenosine receptor present in
membranes isolated from guinea pig and rat cardiac tissue, as
demonstrated by its ability to displace [3H]DPCPX, a
specific adenosine antagonist, from its binding sites. Second, the
cardiac action of the analog is markedly decreased when endogenous
adenosine is removed by pretreatment of atria with ADA, the enzyme that
converts adenosine to inactive inosine. The positive inotropic effect
of the new analog is not affected by the presence of carbachol.
According to Endoh (1979), this result excludes any involvement of cAMP
in the mechanism of cardiotonic action. Moreover, the compound shifts
rightward the cumulative concentration-response curves for the negative
inotropic effect of R-PIA, a stable adenosine receptor agonist. These
cardiac effects, i.e., positive inotropic effect and shift
of R-PIA curves, are more pronounced in rat than in guinea pig atria.
The increase in force of contraction caused by the analog is in fact
45% of Emax in guinea pig atria and 100% of
Emax in rat atria. Furthermore, in guinea pig
atria, 20 µM analog increases the EC50 value of R-PIA
about 2.5 times, whereas in rat atria the same concentration increases
the value 5-fold. It seems reasonable to suggest that the higher
activity of the compound on rat than on guinea pig cardiac tissue is
related to its higher affinity for rat cardiac adenosine receptor. We
calculated Ki values for displacement of [3H]DPCPX of 0.66 ± 0.03 µM and 1.30 ± 0.28 µM in rat and guinea pig cardiac tissue, respectively. In line with
these considerations, it is reasonable to assume that the analog is
more active than milrinone in causing a positive inotropic effect in
both guinea pig and rat atria. In our previous work on guinea pig
cardiac tissue (Dorigo et al., 1992), we calculated a
Ki value of 100 µM for displacement of an
adenosine agonist ([3H]cycloexyladenosine) by milrinone.
In rat cardiac tissue, too, the Ki value for
milrinone is similar (unpublished results). Therefore, the higher
efficacy of the analog with respect to that of the parent compound is
ascribed to its higher ability to displace endogenous adenosine.
The inhibition of soluble Type III PDE is generally considered part of
the mechanism of the cardiac action of milrinone (Endoh et
al., 1986; Weishaar et al., 1986; Silver et
al., 1988; Brunkhorst et al., 1989). In our conditions,
milrinone inhibited guinea pig cardiac Type III PDE in a competitive
way, with a Ki value of 1.4 µM (Dorigo
et al., 1992). Similar behavior and similar kinetic parameters are also evident against rat cardiac Type III PDE (data not
shown). The present data show that the new analog inhibits both guinea
pig and rat cardiac Type III PDE competitively. Dixon plot analysis of
the data yielded Ki values of 11 and 19 µM for Type III PDE isolated from guinea pig and rat heart, respectively. Thus
milrinone, although it is a more potent inhibitor of cardiac Type III
PDE than its analog, is less active as a positive inotropic agent in
both species tested. Moreover, the experiments performed in the
presence of carbachol (Dorigo and Maragno, 1986; present results)
indicate that an increase in cAMP does not play any fundamental role in
the mechanism of action of milrinone and its analog. This suggests that
there is no relationship between the inhibition of cardiac Type III PDE
and the positive inotropic effect of the two drugs. Because some degree
of inhibition of cardiac Type III PDE is always caused by both
milrinone and its analogs, although it is apparently not involved in
their inotropic action, it must be assumed that in rat also (as
documented in guinea pig atria; Weishaar et al., 1987), Type
III PDE may be either biochemically uncoupled from myocardial
contractile proteins or compartmentalized in the cytosol, so that
increases in cAMP concentrations do not influence cardiac
contractility. The present results further support our previous
hypothesis (Dorigo et al., 1992) that milrinone and its
analogs may exert cardiac effects mainly through antagonism toward
adenosine rather than through inhibition of Type III PDE activity.
In conclusion, our data demonstrate that our new synthesized milrinone
analog has higher affinity for cardiac A1 adenosine receptor, and consequently a higher positive inotropic effect, than
milrinone. We are thus encouraged to search for new analogs with even
more pronounced characteristics as antagonists toward endogenous
adenosine, in an attempt to create a safe cardiotonic drug devoid of
any dangerous effect on intracellular cAMP levels. The negative
inotropic effect of adenosine is ascribed to reduced calcium entry into
cells as a consequence of direct K+-channel activation
(Belardinelli and Isenberg, 1983), so an antagonist with high affinity
for the receptor of endogenous adenosine may increase cardiac
contractility without risk of the arrhythmias that always result from
the use of a Type III PDE inhibitor.
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
