(Circulation. 1997;95:2122-2129.)
© 1997 American Heart Association, Inc.
Articles |
`Cross Talk' Between Opioid Peptide and Adrenergic Receptor Signaling in Isolated Rat Heart
From the Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, Baltimore, Md (S.P., R.-P.X., E.G.L.), and the Department of Medical Biochemistry, Ohio State University, Columbus (C.H., R.A.).
Correspondence to Edward G. Lakatta, MD, Chief, Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, 4940 Eastern Ave, Baltimore, MD 21224.
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Background Cardiac myocyte sarcolemma contains both catecholamine and opioid peptide receptors (OPRs). Opioid peptides are coreleased with catecholamines from nerve terminals in the heart. We investigated whether OPR stimulation influences the effects of ß-adrenergic receptor (ß-AR) stimulation in the isolated, isovolumic rat heart and whether the mechanism of such an interaction involves both ß-AR subtypes or an alteration in ß-AR–mediated increase in cAMP.
Methods and Results Norepinephrine (NE,
10-7 mol/L) increased peak left
ventricular systolic pressure (LVSP) and cAMP more
than twofold compared with controls. The 
-OPR agonist
leucine-enkephalin (LE, 10-8 mol/L) markedly
inhibited the ß1-AR–induced positive inotropic effect
and increase in cAMP but alone had no effect on basal LVSP or basal
cAMP levels. The OPR antagonist naloxone
10-8 mol/L added to LE+NE perfusate
reversed the LE-induced decrease in cAMP and LVSP even though naloxone
alone had no effect on LVSP and cAMP levels. LE could not counteract
the twofold increase in LVSP produced by the nondegradable cAMP analog
CPTcAMP 2.3x10-5 mol/L or a high
concentration of forskolin (10-7 mol/L) but
did reverse the 173±11.8% and 135±13.6% increases in LVSP
stimulated by 10-8 and
0.5x10-8 mol/L forskolin, respectively. LE
inhibited cAMP production at all concentrations of forskolin
(10-7, 10-8, and
0.5x10-8 mol/L). Pertussis toxin (PTX)
pretreatment abolished LE effects on ß1-AR stimulation.
Zinterol 10-5 and
10-6 mol/L, a specific ß2-AR
agonist that elicits a cAMP-independent inotropic effect in rat heart,
caused 225±14% and 182±5% increases in LVSP that could not be
reversed by addition of LE.
Conclusions Potent, inhibitory "cross
talk" between -OPR and ß1-AR signaling pathways
occurs via a PTX-sensitive Gi/o protein involved in
adenylyl cyclase inhibition in rat heart.
Key Words: opioid peptides • receptors, adrenergic, beta • cAMP • proteins • rats
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Introduction |
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After sympathetic stimulation, OPs and catecholamines are coreleased from neuronal terminals in heart tissue.1 Arterial tonus, arterial pressure, heart rate, and force of contraction are affected by OP.1 In the past, these effects have been attributed solely to a presynaptic role of OP modulation of catecholamine release from autonomic nerve endings in the heart and vasculature.1 However, more recently it has been shown that OPs are also produced by cardiac myocytes2 3 4 5 6 and that cardiac ventricular sarcolemma contains functional OPRs.7 8 9 10 11 12 Although OPR stimulation decreases cAMP levels in the rat heart,13 14 it is still unclear whether such an OPR-stimulated decrease in cAMP is secondary to a presynaptic action of OPs that leads to a reduced corelease of catecholamines and subsequently reduced ß-AR stimulation or whether there is a postsynaptic intracellular signaling mechanism of interaction that modulates the positive inotropic effect and increased cytosolic cAMP levels induced by ß-AR stimulation. Although it has long been shown in brain that OPs have no effect on basal cAMP levels while markedly inhibiting NE-stimulated cAMP formation,15 this OPR-stimulated "cross talk" with the ß-AR signaling pathway is relatively unexplored in heart.
The specific purpose of this study was to test whether and how -OPR
stimulation by LE alters the modulatory effects of ß1-AR
stimulation to increase cAMP and contraction in isolated rat hearts in
which catecholamines had been depleted from nerve endings.
We recently confirmed that the effects of ß1-AR
stimulation to increase cytosolic Ca2+ transients and
contraction amplitude of rat ventricular myocytes are
tightly coupled to increases in cAMP.16 However, the
effects of ß2-AR stimulation on
contractility and cytosolic Ca2+ transients
in this cell type16 and in dog ventricular
cells are dissociated from an increase in cellular cAMP
levels.17 In a separate subset of hearts, we used the
specific ß2-AR agonist zinterol to test whether LE had
differing modulatory effects on ß-AR subtype actions. In an
additional group, the ability of LE to alter the increase in
contraction and cellular cAMP after the stimulation of adenylyl cyclase
by forskolin was tested.
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Methods |
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Isolated Heart Preparation
Male Sprague-Dawley rats (300 to 400 g) were used for standard isolated isovolumic heart perfusions. All animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals. Rats were injected with 6-OHDA 20 mg/kg IP 24 hours before isolated heart perfusions, which we have shown previously to deplete
80% to 90% of the
catecholamine content of peripheral nerve
endings.3 Pretreatment with 6-OHDA was used to minimize
neural catecholamine influence on the isolated
myocardium. However, no significant difference in LVSP and
coronary flow was observed between hearts from 6-OHDA–treated
and –untreated rats for control buffer perfusion, NE, NE+LE, and
NE+LE+NAL (n=10). All data presented in this study are from
6-OHDA–pretreated hearts. After rapid cannulation of the aorta,
coronary perfusion was commenced at a constant pressure of
85 mm Hg with a filtered (0.45 µm) bicarbonate buffer.
This consisted of (in mmol/L) KCl 3.48, NaCl 116.4,
NaHCO3 26.2, NaH2PO4 1.67,
MgSO4 0.69, CaCl2 1.5, and glucose 11.1. The
perfusate was gassed with 95% O2/5%
CO2 and equilibrated at pH 7.38 to 7.42. Temperature was
maintained at 30°C to prevent the occurrence of arrhythmias
during perfusion with NE, zinterol, or forskolin at concentrations that
produced a greater than twofold increase in peak LVSP. The
coronary flow rate, which was monitored during all experimental
protocols, was maintained at 19±0.5 mL/min, and no significant
fluctuations in coronary flow occurred during any drug
treatment. Hearts were electrically paced at 3 Hz.
Ventricular function was assessed by measurement of LVSP
with a fluid-filled latex balloon inserted via the left atrium and
connected via a polyethylene catheter to a pressure transducer (Gould
model P23GB). Developed pressure was recorded on a Gould model
2400S chart recorder. The balloon was inflated to yield a left
ventricular end-diastolic pressure <8
mm Hg. Perfusion of a subset of hearts (n=4) was monitored for a total
of 3 hours to establish the stability of the preparation well beyond
the period of experimental protocols. After 3 hours of control
perfusion, peak systolic pressure did not decline significantly
(92±12%, mean±SEM of initial control).
Experimental Protocol
After control perfusion equilibration (10 to 20 minutes), NE
10-7 mol/L was administered. This
concentration was selected to invoke a twofold increase in peak
systolic pressure and is also the concentration at which the
maximal increase in LVSP can be measured with sustained stability.
Higher doses of NE, 10-6 mol/L, led to
arrhythmias and decreased performance stability,
preventing unconfounded measurement of LVSP in this preparation. When
the NE-induced rise in peak systolic pressure had stabilized
for 10 to 20 minutes, LE 10-8 mol/L was added
with NE. After stabilization of an LE effect (15 to 25 minutes), NAL
10-8 mol/L was added in the continued presence
of LE and NE. A subset of isolated hearts were perfused with either LE
10-8 mol/L or NAL
10-8 mol/L alone.
To assess a possible role of inhibitory G proteins (Gi/Go) in LE actions, rats were injected with PTX 25 µg/kg IP 24 hours before isolated heart perfusion. Successful inactivation of inhibitory G proteins in isolated hearts was verified by a loss of the efficacy of adenosine 10-6 mol/L to reverse the positive inotropic effect of NE. The above protocols were repeated in PTX-pretreated hearts.
In other experiments, at doses equipotent to NE, either zinterol 10-5 or 10-6 mol/L, CTPcAMP 2.3x10-5 mol/L, or forskolin 10-7, 10-8, or 0.5x10-8 mol/L was used to stimulate an increase in the average peak systolic pressure. After stabilization, LE 10-8 mol/L was perfused to test whether it affected the respective stimulated increases in systolic pressure.
Cardiac ventricles were freeze-clamped in liquid nitrogen after
perfused hearts had equilibrated in steady state according to their
respective drug treatment. This steady-state period corresponded to the
assessment period (
15 minutes) for average peak systolic
pressure. Frozen tissue wafers were homogenized under
liquid nitrogen for preparation of neutral extracts in which cAMP
content was analyzed by radioimmunoassay.18
Drugs
NE, adenosine, PTX, forskolin, and CPTcAMP were
obtained from Sigma Chemical Co. Zinterol was kindly supplied by
Bristol-Myers and ICI 118,551 by Imperial Chemical Industry.
Statistics
Data are reported as mean±SEM. Statistical comparisons were
made by one-way ANOVA, repeated-measures ANOVA, and Tukey's
studentized range (honest significant difference) test where
appropriate. A value of P<.05 was considered to be
statistically significant.
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Results |
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Effects of LE on ß1-AR Signal Transduction Pathway
Fig 1A
illustrates an
intraventricular pressure tracing from a
representative isovolumic rat heart preparation. A
greater than twofold increase in peak LVSP occurs in response to NE
10-7 mol/L. We have previously shown that NE
acts predominantly as a ß1-AR agonist in isolated rat
cardiac myocytes,19 because prazosin (an
1-adrenergic antagonist;
10-6 mol/L) exerted no significant alteration
of the effects of NE on rat myocyte intracellular calcium transient and
contraction.19 20 The average NE-induced increase of LVSP
was 229±12 mm Hg (Fig 1B). LE 10-8
mol/L in the continued presence of NE reversed the NE effect (example
shown in Fig 1A; average effect shown in Fig 1B). Pilot experiments,
however, showed that higher concentrations of LE alone,
10-7 and 10-6 mol/L,
significantly reduced LVSP to 67.9±7.5 and 33.5±5 mm Hg,
respectively (n=3). A similar negative inotropic effect occurs in
isolated rat ventricular myocytes.10 Although
LE 10-8 mol/L markedly inhibited the positive
inotropic effect of NE, when added to the perfusate in the
absence of NE, it had no significant effect on LVSP (Fig 1B). NAL
10-8 mol/L, an OPR antagonist, in
the continued presence of LE and NE reversed the effect of LE (example
in Fig 1A; average effect in Fig 1B). NAL 10-8
mol/L alone had no significant effect on cardiac contraction (Fig 1B).
Results of additional experiments in hearts not pretreated with 6-OHDA
did not differ from those in Fig 1.
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P<.05 vs LE+NE; P=NS
for LE+NE vs LE alone and for LE+NE vs C. Open bars indicate hearts
perfused with either LE 10-8 mol/L or NAL
10-8 mol/L alone for a total of 60 minutes.
P=NS for LE 10-8 mol/L or
NAL10-8 mol/L alone vs C.
The concentration of cAMP in ventricular tissue was
directly determined for each of the above treatments (Fig 2). After NE 10-7 mol/L, the
increase in LVSP was associated with a greater than twofold increase in
cAMP. Perfusion with LE 10-8 mol/L in the
presence of NE caused a significant decrease in cAMP; however, this
concentration of LE when perfused alone did not affect the cAMP level.
NAL 10-8 mol/L alone had no significant effect
on basal cAMP levels, but when added after LE+NE, the LE-induced
decrease in NE-raised cAMP was significantly reversed (Fig 2).
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LE Does Not Reverse ß2-AR–Stimulated Rise in
Developed Pressure
Although both ß1- and ß2-ARs are
coupled to adenylyl cyclase via Gs,
ß1-AR but not ß2-AR stimulation leads to
protein kinase A–mediated increases in cardiac myocyte phospholamban
phosphorylation in dog and rat ventricular
cells.16 17 In dog cells, ß2-AR does not
increase cAMP,17 21 and in rat cells, the increase in
contraction amplitude after ß2-AR stimulation is
dissociated from its effect to increase cAMP.16 22 To test
the effects of LE on a ß2-AR–mediated increase in LVSP,
the highly specific ß2-AR agonist zinterol
10-5 or 10-6 mol/L
was used, which produced 225±14% and 182±5% increases in LVSP,
respectively (n=4). A representative example of the
zinterol effect is shown in Fig 3A, and the average
zinterol-induced rise in LVSP is shown in Fig 3B. LE
10-8 mol/L did not reverse the positive
inotropic effect of 10-5 or
10-6 mol/L zinterol. However, the specific
ß2-AR antagonist ICI 118,551
10-7 mol/L in the presence of zinterol
10-5 mol/L and LE
10-8 mol/L completely reversed the positive
inotropic effect of zinterol (98±8% of control, n=3,
P<.001).
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Effect of LE on CPTcAMP- or Forskolin-Mediated Rise in
Developed Pressure
Specific effects of LE on the ß-AR signaling cascade to reverse
the ß1-AR agonist–mediated increase in LVSP and
cytosolic cAMP could be possible by an interaction at a number of sites
along the ß1-AR signal transduction pathway (see Fig 4). LE could possibly reduce the coupling of the
ß1-AR to Gs or Gs to the
catalytic subunit of adenylyl cyclase, inhibit adenylyl cyclase via
Gi/o protein coupling, or increase phosphodiesterase
activity and the degradation of cAMP. To directly increase
intracellular cAMP without activating the
ß1-AR/Gs complex or subsequent
Gs–adenylyl cyclase interaction or adenylyl
cyclase itself, CPTcAMP, a synthetic nondegradable analogue of cAMP,
was used. The concentration of 2.3x10-5 mol/L
CPTcAMP was selected to produce an approximately twofold rise in LVSP
and was shown to be the EC50 in pilot experiments with
isolated ventricular myocytes (R.-P.X. et al, unpublished
data). Perfusion with CPTcAMP increased LVSP to 176±14% (n=5) of
control (Fig 5A presents a
representative example; Fig 5B shows average data). LE
10-8 mol/L failed to inhibit the approximately
twofold increase in LVSP induced by 3x10-5
mol/L CPTcAMP.
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Forskolin, an activator of adenylyl cyclase, at
10-7 mol/L, a concentration equipotent to that
of NE in augmenting systolic pressure, significantly raised
LVSP to 246±13.7% (n=4) of control levels but was not affected by the
addition of 10-8 mol/L LE (Fig 6). However, the marked forskolin-induced
10-7 mol/L increase in cAMP concentration to
602±18.5% of control was significantly reduced by
10-8 mol/L LE. Because the residual cAMP
induced by 10-7 mol/L forskolin plus
10-8 mol/L LE was significantly greater than
the rise in cAMP induced by 10-7 mol/L NE (Fig 2
), we also used lower concentrations of forskolin. The rise in LVSP
produced by 10-8 mol/L forskolin (173±11.8%
of control, n=4) or 0.5x10-8 mol/L forskolin
(135±13.6% of control, n=4) was reduced significantly, to 110±13.6%
and 105±2.7%, respectively, by 10-8 mol/L
LE. In these hearts, the concentration of cAMP was reduced from
397±19.8% to 94±5.8% of control and from 139±8.5% to 102±2.8%
of control in the presence of 10-8 or
0.5x10-8 mol/L forskolin, respectively. Thus,
because of the increases in cAMP of 600% and 400% of control induced
by 10-7 and 10-8
mol/L forskolin, respectively, the relationship of the
forskolin-induced increase in LVSP and cAMP (Fig 6) is nonlinear, as
previously shown.18 23 The -OPR–induced alteration of
the low forskolin concentration and ß1-AR–stimulated
increase in LVSP was found to have a close correlation with the
increase in cAMP concentration, as determined by linear regression (Fig 7), which occurred over a cAMP concentration range up to
200% of control. LE antagonized the cAMP increased by forskolin at
all concentrations of the latter but reduced the forskolin-induced
increase in LVSP only along the linear portion of the [cAMP]-LVSP
relationship.
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, NE 10-7 mol/L;
, NE+LE; 
, NE+LE+NAL;
, NAL 10-8 mol/L; 
, PTX:NE+LE;
*, forskolin 10-7 mol/L; 
, forskolin
10-7 mol/L+LE 10-8
mol/L; 
, forskolin 10-8 mol/L; 
,
forskolin 10-8 mol/L+LE
10-8 mol/L; 
, forskolin
0.5x10-8 mol/L; and 
, forskolin
0.5x10-8 mol/L+LE
10-8 mol/L. Forskolin
10-7 (*) and
10-8 mol/L (
LE-NE Interaction Occurs Via a PTX-Sensitive Gi/o
Protein
The -OPR–stimulated decrease in cAMP after ß1-AR
stimulation in heart may occur as a consequence of -OPR coupling to
an inhibitory G protein, Gi/o, that inhibits
adenylyl cyclase activity. To test whether LE couples to
Gi/o, 24 hours before hearts were harvested, rats were
treated with PTX 25 µg/kg IP, which catalyzes the adenine
nucleotide ribosylation of Gi/o protein
-subunits and inhibits the response to Gi/o-coupled
receptor agonists. Fig 8A shows the effect, in a typical
isolated heart, of adenosine 10-6
mol/L to reverse the NE-stimulated increase in LVSP. The average effect
of adenosine 10-6 mol/L+NE
10-7 mol/L in three hearts was to reduce
developed pressure to 98±5% of control from 224±15% during
perfusion with NE alone (P<.01). After PTX pretreatment,
the adenosine effect was abolished (Fig 8B). Pretreatment with
PTX did not affect LVSP achieved during perfusion with control buffer
(102±4% of non–PTX-pretreated control) or with NE (217±14% of
PTX-treated control) but blocked the average effect of
adenosine (99±5% of PTX-treated control) (n=8). Pretreatment
with PTX prevented LE inhibition of the positive inotropic effect and
the increase in cAMP induced by NE (Figs 3 and 8). Thus, the inability
of LE 10-8 mol/L to reverse NE-augmented LVSP
after PTX pretreatment indicates that the OPR pathway interacts with
the ß1-AR pathway via a PTX-sensitive G protein–mediated
mechanism (Figs 4 and 9). Although LE appears to act
like adenosine, we noted a difference in the time course
between the reversal of NE-stimulated inotropy by adenosine
(1.6±0.2 minutes, n=3) and LE (16.5±2.3 minutes, n=10;
P<.01).
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Discussion |
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The present results indicate, for the first time, that a marked antiadrenergic effect of LE in the isolated rat heart is due to a specific postsynaptic intracellular action of LE on the ß1-AR stimulation pathway via a PTX-sensitive G protein that is involved in the regulation of cell cAMP levels. Furthermore, in our preliminary studies using isolated rat ventricular cardiac myocytes, LE 10-8 mol/L reversed ß1-AR–stimulated increases in Ca2+ transient and contraction amplitude, which could be blocked by cell pretreatment with PTX.24 Specifically, the present results in 6-OHDA–pretreated, intact isolated rat hearts show that the ß1-AR–stimulated increases in peak systolic pressure and cAMP were markedly reversed by LE at a concentration of the latter that alone had no significant influence on these parameters. We believe that these effects occur postsynaptically at the level of the cardiac myocyte because nerve terminals were blocked by 6-OHDA pretreatment and there was no change in coronary flow. The fact that LE could not antagonize a similar increase in LVSP evoked by CPTcAMP strongly suggests that the effect of LE was mediated by a reduction in cAMP. The LE-induced reversal of the effect of forskolin, at concentrations of the latter that produced an increase in cAMP in the range in which LVSP varies with cAMP (Fig 7
), indicates that the LE effect occurs distal to the
ß1-AR–Gs
coupling.25 26 The fact that the LE counteraction of
ß1-AR–stimulated increase in LVSP and cAMP can be
abolished by pretreatment with PTX (which catalyzes the adenine
nucleotide ribosylation of Gi/Go
protein -subunits and inhibits the response to
agonists27 ) may be explained either by an effect of
Gi/o coupling to the -OPR to directly inhibit adenylyl
cyclase or to stimulate phosphodiesterase28 (Fig 4).
Although it has been reported that -OPR activation results in
adenylyl cyclase inhibition via coupling to
Gi2, Gi3, and
Go in neuroblastomaxglioma hybrid cells
(NG108-15)29 30 and
Gi1/Go1 in human
neuroblastoma cells (SH-SY5Y),31 a similar effect of
-OPR to decrease adenylyl cyclase has not as yet been shown to occur
in heart cells. The present study cannot discriminate among which
of the five PTX-sensitive inhibitory G-protein
subtypes27 are coupled to the cardiac -OPR.
The ß2-AR specific agonist zinterol at
10-5 and 10-6 mol/L
increased LVSP to 225±14% and 182±5% of control, but LE
10-8 mol/L failed to counter the effects of
both concentrations of zinterol, in contrast to the effect of LE on the
ß1-AR signaling pathway. The specific ß2-AR
antagonist ICI 118,551 completely blocked the positive
inotropic effect of zinterol (Fig 3A) and reduced LVSP to 98±8% of
control. Zinterol (including 10-5 mol/L)
displacement of (-)-125I-cyanopindolol binding and
zinterol-induced positive inotropy have been shown to be unaffected by
the highly selective ß1-AR antagonist CGP
20712A but blocked by ICI 118,551,32 thus clearly
indicating that zinterol at the doses we used has no significant
ß1-AR activity. The present study findings were
predicted in view of Fig 4 because, although both ß1- and
ß2-ARs are coupled to a Gs and
increase cAMP formation in rat heart, ß2-AR
stimulation–induced increase in rat cardiac myocyte contraction
amplitude is dissociated from the cAMP-dependent protein kinase A
pathway.16 Notably, ß2-AR stimulation in
canine cells does not even increase cAMP.17 Rather, in
both species, the effects of ß2-AR stimulation to
increase [Ca2+]i and contraction are due to
an increase in L-type Ca2+ current amplitude, perhaps via a
direct G-protein coupling rather than cAMP-related Ca2+
channel protein
phosphorylation.16 17 19 21 22
In single cardiac myocytes, OPs have been shown to increase
IP3 and affect protein kinase C–dependent
mechanisms7 11 (see Fig 4). In noncardiac hybrid cell
lines, -OPR stimulation also mediates the activation of
phospholipase C for phosphoinositide
metabolism and formation of
IP3.33 34 In cardiac cells, in the absence of
an increase in Ca2+ influx, IP3 depletes the
sarcoplasmic reticulum of Ca2+ and decreases
contraction.35 An increase in protein kinase C activity
has also been indirectly shown to decrease twitch amplitude and calcium
transient in rat cardiac myocytes.35 Although activation
of the IP3-forming pathway has been attributed to
PTX-insensitive Gq protein36
and Gi/oß
,37
specific coupling of Gq and/or Gi/o to cardiac
-OPR awaits direct demonstration. These effects of OPR activation on
IP3 and cAMP formation are suggestive of a potential dual
or multiple G-protein coupling. However, under baseline conditions at
least, the negative inotropy that may be induced by OPR
stimulation7 11 was obviated in the present study by
use of a concentration of LE, 10-8 mol/L, that
had no appreciable effect on basal LVSP or cAMP levels (Figs 1B and 2).
Thus, the lack of an effect of 10-8 mol/L LE
alone would be consistent with previous reports that OPR
activation in brain or heart tissue results in decreased cAMP levels
but that the extent to which this can occur is determined by the levels
of prior adenylyl cyclase activity and cAMP
formed.13 14 38 39
It is noteworthy that in the present study, LE was unable to
inhibit the increase in LVSP induced by 10-7
mol/L forskolin but did significantly reduce the forskolin-stimulated
increase in cAMP. This supports the notion that at high concentrations
of cAMP, the relationship between cAMP and developed pressure becomes
nonlinear18 23 (Figs 6 and 7). The present study and
those of others18 23 have shown that forskolin is more
effective in raising intracellular cAMP than ß1-AR
stimulation for the same increase in contraction. When lower
concentrations of forskolin were used (10-8 or
0.5x10-8 mol/L), LE
10-8 mol/L reversed forskolin-stimulated
increases in both LVSP and cAMP. Thus, after NE+LE, cAMP levels need to
decrease below a specific "threshold," ie, to less than
approximately twofold over baseline, where the relationship between
cAMP and LVSP is linear, before ventricular contraction
amplitude becomes reduced. The finding that LE
10-8 mol/L does not inhibit the positive
inotropic action of cAMP itself (without the activation of adenylyl
cyclase), ie, via CPTcAMP, suggests that LE may not act downstream from
the activation of cAMP-dependent protein kinase A. However, because
CPTcAMP is a nondegradable analogue of cAMP, the possibility that LE
acts to inhibit forskolin or ß1-AR–stimulated increases
in cAMP via an increased activation of phosphodiesterase28
cannot be ruled out (see Fig 4).
The "cross talk" between cardiac -OPR and ß1-AR
signal transduction pathways demonstrated by the present results
may thus govern the magnitude of ß1-adrenergic modulation
of cardiac excitation-contraction coupling (see Fig 4), as has also
been shown for other PTX-sensitive Gi/o protein–coupled
receptors, such as adenosine,40 41 muscarinic
acetylcholine receptor,40 42 or ET
receptors.43 44 45 46 All of these receptor types
simultaneously activate multiple transduction
pathways, which, like OPR,24 25 30 31 33 involve both
cAMP- and IP3-forming pathways.43 44 45 46 47 48 Despite
similarities between these receptor-coupled transduction systems,
different specific mechanisms or condition-dependent circumstances may
contribute to differences in their efficacy and time course of
interaction with the ß1-AR pathway in situ or to the
relevance of the latter. Notably, in the present study, although
both LE and adenosine each effectively countered NE-stimulated
inotropy, there was a disparity in the time course of reversal by
adenosine (1.6±0.2 minutes) and LE (16.5±2.3 minutes),
perhaps related to differences in the localization, number, and
affinity of each receptor type7 40 or to differences in
the interaction of their signaling cascades with that coupled to the
ß1-AR. During conditions of ischemia or
hypoxia, adenosine production is markedly
increased49 and ETA receptor density is
rapidly increased, whereas elevated catecholamine levels
downregulate ETB receptors.50 At rest, when
cardiac work and catecholamines are very low, the cross
talk between muscarinic acetylcholine receptor and ß1-AR
signaling pathways is predominant. However, with increased cardiac work
at high catecholamine levels, the adrenergic-cholinergic
interaction is blunted and -OPR pathway interactions with the
ß1-AR pathway would be expected to be increased according
to opioid and catecholamine corelease.1
Another interaction between the opioid and adrenergic pathways at the
level of OPR agonist binding may also occur when NE and OPs are
coreleased from nerve endings, because OPR ligand binding to cardiac
cell sarcolemma has been shown to be increased in the presence of
catecholamines.7 These results suggest that
under basal conditions, OPRs may not be abundantly available but that a
rise in catecholamines prompts a significant increase in
the number and affinity of 
- and -OPRs.7
During periods of cardiac stress when ß-AR stimulation increases cAMP
above basal levels, an OPR-stimulated attenuation of cAMP, particularly
in response to increased metabolic substrate demand during
intense exercise or cardiac ischemia, might be expected to
prevent metabolic demand from exceeding supply of substrate
and limit calcium overload and calcium-dependent arrhythmias.
In two recent preliminary studies, preconditioning or OPR stimulation
by an opioid agonist could equivalently abolish the
ventricular tachycardia and fibrillation
induced by brief coronary artery ischemia and
reperfusion51 and significantly reduced infarct size
compared with controls.52 In addition, we have previously
shown that OPs and their preproenkephalin mRNA are both markedly
increased in rat hearts with adult aging.2 3 This may be a
contributing factor to the reduction in ß1-AR–mediated
inotropic response observed with aging,20 particularly
because cardiac myocytes secrete OPs.4 Preproenkephalin
mRNA levels are also significantly elevated in hearts from the
cardiomyopathic hamster strain53 and
spontaneously hypertensive rat strain.54 Recent data also
suggest that in congestive heart failure, elevated
endogenous opioids may act via -OPRs to decrease cardiac
mechanical performance.55 Although the
significance of the findings from these studies is yet to be
established, it is tempting to predict a compensatory protective role
for increased OP-mediated regulation.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was funded in part by NIH grant HL-48835. We thank Lourdes Castillo for performing cAMP assays.
Received June 25, 1996; revision received October 23, 1996; accepted November 19, 1996.
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References |
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