© 1995 American Heart Association, Inc.
Articles |
ß2-Adrenergic Receptor Actions in Neonatal and Adult Rat Ventricular Myocytes
From the Departments of Medicine (S.F.S.) and Pharmacology (V.K., E.P., R.B.R., S.F.S.), Columbia University, New York, NY.
Correspondence to Susan F. Steinberg, MD, Associate Professor of Medicine and Pharmacology, Department of Medicine, Columbia University, College of Physicians and Surgeons, 630 West 168 St, New York, NY 10032.
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Abstract The physiological function of ß2-adrenergic receptors in the neonatal and adult heart is incompletely understood, and possible age-dependent differences in ß2-receptor actions have not been considered. We used isoproterenol (mixed ß1- and ß2-receptor agonist) and zinterol (ß2-selective agonist) to compare ß-receptor subtype actions in neonatal and adult rat ventricular myocytes. When delivered as a bolus at a final concentration of 10-7 mol/L, both isoproterenol and zinterol increased the amplitude and hastened the kinetics of the calcium and cell-shortening transients in neonatal myocytes. Under identical experimental conditions, isoproterenol increased the amplitude and accelerated the kinetics of the calcium transient and the twitch in adult myocytes, whereas zinterol did not. In the presence of CGP 20712A (ß1-receptor blocker), a 100-fold higher concentration of zinterol increased the amplitude but prolonged the duration of the twitch in adult myocytes. To probe the mechanism for this age-dependent difference in ß2-receptor responsiveness, we compared ß-receptor expression and stimulation of cAMP accumulation in neonatal and adult myocytes. ß-Receptor density was 44 339±5178 sites per cell in neonatal myocytes and 186 346±13 356 sites per cell in adult myocytes; the relative proportion of ß2-receptors was comparable in each (16.7±2.3% and 16.9±0.9%, respectively). Isoproterenol induced a large increase in cAMP accumulation in neonatal and adult myocytes (20.0±1.0- and 20.6±2.6-fold over basal). In contrast, zinterol evoked a substantial increase in cAMP accumulation in neonatal myocytes but only a minor increase in adult myocytes. These studies provide evidence that at low agonist concentrations, ß2-receptor activation contributes to the positive inotropic response by increasing cAMP and increasing the amplitude and hastening the kinetics of the twitch in neonatal, but not adult, myocytes. Moreover, these results suggest that age-dependent differences in ß2-receptor coupling to more distal elements in the signaling cascade can influence myocyte ß2-receptor responsiveness.
Key Words: ß-adrenergic receptor subtypes • cardiac myocytes • development • Ca2+ • contraction
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Introduction |
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It is generally accepted that under physiological conditions catecholamines exert positive chronotropic and inotropic responses through the activation of the more abundant cardiac ß1-adrenergic receptor subtype, which couples to the stimulation of adenylyl cyclase activity, the accumulation of cAMP, and cAMP-dependent phosphorylation of numerous intracellular target-regulatory proteins. However, radioligand binding studies provide evidence that ß1- and ß2-adrenergic receptors coexist in the hearts of several mammalian species (Reference 11 and references cited therein). Although ß2-adrenergic receptors also couple to the stimulation of adenylyl cyclase and the generation of cAMP, the functional role of cardiac ß2-adrenergic receptors in the modulation of cardiac contractile function has remained somewhat obscure.
In the human heart, ß2-adrenergic agonists have been shown to induce tachycardia by stimulating ß2-adrenergic receptors in the sinoatrial node.2 Similarly, there is convincing data that ß2-receptors couple to a positive inotropic response in human ventricular myocardium.3 However, there are discrepancies between the effect of ß2-receptor agonists to stimulate adenylyl cyclase and to modulate contractile function, raising questions regarding the precise relation between ß2-receptor–dependent stimulation of cAMP accumulation and modulation of contractile function.3 4 The literature defining the role of ß2-adrenergic receptors in the contractile response to catecholamines in nonhuman cardiac tissues also leaves many questions unanswered. Results of some studies lend support to the notion that selective ß2-adrenergic receptor agonists have a greater effect on heart rate than on contractility.5 6 Nevertheless, ß2-adrenergic receptors have been localized by quantitative autoradiography to both the conduction system and the working myocardium7 8 9 of the dog and guinea pig heart and have been reported to contribute to both the positive chronotropic and inotropic responses to catecholamines in these species.6 10 11 12 In contrast, the evidence for significant modulation of the contractile properties of the rat heart by ß2-receptor agonists is less convincing. For example, Juberg et al13 concluded that despite the presence of ß2-adrenergic receptors in rat atrial membrane preparations, only ß1-adrenergic receptors mediate the positive chronotropic response in spontaneously beating right atria or the positive inotropic response in electrically driven left atria. Effects of ß2-receptor agonists to increase the contractile rate subsequently have been identified. However, it is noteworthy that the authors of these studies have emphasized that the ß2-receptor effect is a minor component of the chronotropic response, which only comes into play at very high catecholamine concentrations.14 15 Similarly, although a more recent study describes an effect of ß2 agonists to increase the amplitude of the twitch and calcium transient in isolated adult rat ventricular myocytes,16 this ß2-receptor response also required a high concentration of agonist. Nevertheless, it is noteworthy that this study uncovered intriguing differences in several aspects of the cellular response to ß1- and ß2-adrenergic receptor agonists, leading the authors to conclude that the mechanism(s) whereby cardiac ß1- and ß2-adrenergic receptors modulate contractile function must differ.
Studies of ß-receptor subtype action in rat heart to date have been performed exclusively on adult tissue. Although both ß1- and ß2-adrenergic receptors have been detected by radioligand binding techniques in neonatal rat ventricular membranes,17 the role of a ß2-receptor signaling pathway in the neonatal heart has never been considered. Therefore, the objectives of the present study were to determine whether ß2-receptors are important mediators of inotropic support in the neonatal heart and to investigate the role of cAMP in the ß2-adrenergic receptor response.
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Preparation of Acutely Disaggregated Adult and Cultured Neonatal Rat Ventricular Myocytes
Cardiac myocytes were isolated from hearts of 2-day-old Wistar rats by a trypsin dispersion procedure and cultured for 5 days according to a protocol described previously.18 Measurements of cell shortening and cytosolic calcium were performed on myocytes grown on round fibronectin-coated glass coverslips (0.1-mm thickness, 31-mm diameter; Biophysica Technologies, Inc) in 35-mm culture dishes. Although the culture technique includes a preplating step that effectively decreases fibroblast contamination, it is well known that a small number of cells with proliferative capability, such as cardiac fibroblasts, persist in the myocardial cell cultures. Proliferation of these cells was further curtailed with 0.1 mmol/L bromodeoxyuridine (present in the culture medium from days 1 through 519 ) for calcium and contraction studies and with an irradiation protocol20 for biochemical studies.
Adult rat ventricular myocytes were disaggregated according to methods
published previously with minor modifications.18 Male
Wistar rats weighing 300 to 350 g (
10 to 12 weeks old) were
anesthetized and heparinized before removal of the heart. The aorta was
cannulated, and the heart was perfused with 20 to 25 mL of low-calcium
(2 µmol/L, from calcium pantothenate) HEPES buffer (mmol/L: NaCl 118,
KCl 5.6, NaHCO3 4.4, NaH2PO4 1.74,
MgCl2 1.69, glucose 5.6, L-glutamine 4.3,
HEPES 21, and taurine 10, along with 20 mL/L MEM amino acid solution
and 10 mL/L MEM vitamin solution; pH 7.2; osmolarity, 295 mosm),
followed by perfusion for 10 to 15 minutes at 35°C with the same
HEPES buffer containing 0.37 mg/mL collagenase (Worthington CLS II).
The perfusion was terminated when the heart became soft. The ventricles
were then isolated and placed in a flask containing 10 mL of the same
buffer with 1.1 mg/mL collagenase, 0.6 mmol/L CaCl2,
and 0.5% bovine serum albumin (BSA, salt-free fraction V from ICN).
The flask was shaken vigorously for 10 to 30 minutes at 32°C. Cells
in the supernatant were collected, filtered through the polyethylene
macrofilter, centrifuged at 600 rpm for 3 minutes, and resuspended in
10 mL HEPES buffer containing 1 mmol/L CaCl2 and 0.5% BSA.
This procedure was repeated three times, and cells from each digest
were stored separately. Cells from 17 separate adult ventricular
myocyte preparations were used in this study. Cells were stored at room
temperature in HEPES buffer containing 1 mmol/L CaCl2 and
0.5% BSA and used within 1 to 10 hours after preparation for
measurements of cytosolic free calcium, cell shortening, and
agonist-stimulated cAMP accumulation. Cells were frozen at -70°C in
HEPES buffer containing 1 mmol/L CaCl2 and 0.5% BSA at a
density of 105 cells per milliliter for radioligand binding
experiments. Broken adult rat ventricular myocytes prepared in this
manner were used within 1 week of freezing.
Measurement of Cytosolic Free Calcium and Cell Shortening
Methods for the photometric measurement of cytosolic calcium in
fura 2–loaded cultured neonatal and acutely disaggregated adult
myocytes have been published previously.20 In brief,
myocytes were loaded with the acetoxymethyl ester form of fura 2 by
incubating neonatal myocyte monolayer cultures on a coverslip or
100 000 adult myocytes in suspension with 3 µmol/L fura 2-AM and 1.5
µL of 25% (wt/wt in dimethyl sulfoxide) Pluronic F-127 (BASF
Wyandotte Corp) dissolved in 1.0 mL Tyrode's solution for 20 minutes
at 37°C. Myocytes were rinsed with fresh Tyrode's solution and
maintained for at least 15 minutes at room temperature to allow for
deesterification of the dye.
The device used for monitoring the fluorescence of intracellular fura 2 (Photon Technologies, Inc) alternately illuminates the cells with 340- and 380-nm light while measuring emission at 520 nm. Sampling rate for collection of ratio values is 100 Hz. Cytosolic free calcium ion concentration theoretically can be calculated from the fura 2 fluorescence ratio at the two excitation wavelengths. Although numerous approaches to calibrate intracellular fura 2 have been presented, it is extremely difficult to entirely circumvent uncertainties in the calibration because of potential compartmentalization of the dye in fura 2-AM–loaded cells, differences in the spectral properties of fura 2 in cells and in buffer solutions, and fluorescence changes due to cell contracture during calibration protocols.21 22 Because of these unavoidable uncertainties and because fura 2 fluorescence provides an extremely sensitive indicator of changes in intracellular calcium during experimental protocols (the primary focus of these studies), we have reported intracellular calcium as the fura 2 fluorescence ratio.
To monitor cell motion, the cells were simultaneously illuminated with red light, and a dichroic mirror (630-nm cutoff) in the emission path deflected the cell image to a video optical system (Crescent Electronics) that tracked the motion of the cell ends (adult myocytes) or a high contrast microsphere attached to the myocyte surface (neonatal myocyte, see below) along a raster line segment of the image during electrically stimulated contractions. The analog voltage output from the motion detector was calibrated to convert to micrometers of motion. The motion signal was obtained at a rate of 60 Hz and reflected the motion of the same myocyte simultaneously monitored with fura 2 for calcium. The signal was digitized and stored along with the fluorescence data.
Glass beads (2.1±0.5 µm, Duke Scientific Corp) were added to the
neonatal monolayer cultures to provide high-contrast spots for tracking
cell motion. Measurements of changes in cell length are not possible,
because these cells lack a single axis of myofibrillar alignment.
Therefore, studies of individual cultured neonatal myocytes report cell
shortening. In contrast, the rod-shaped geometry of adult ventricular
myocytes permits monitoring of actual cell length. In this case, the
relative positions of the ends of the long axis of the cell were
tracked. These differences in the methods used to measure motion in
neonatal and adult ventricular myocytes preclude a detailed comparison
of the average amplitude of cell shortening during the twitch between
neonatal and adult myocytes. However, measurements of the kinetics of
the calcium transient and the twitch in neonatal and adult rat
ventricular myocytes are possible. As illustrated in Figs 1
and 2, the
kinetics of the calcium transient and cell shortening are considerably
more rapid in the adult myocyte compared with the neonatal myocyte.
This marked age-dependent difference in the baseline kinetics of the
calcium transient and contraction is similar to that previously
documented in papillary muscle.23
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Studies were performed by using a three-compartment superfusion chamber
modified so that a coverslip formed the bottom of the chamber, as
described previously.20 The cells were superfused with
Tyrode's solution gassed with 95% O2/5%
CO2 at a rate of 1 mL/min. Experiments were performed at
room temperature. Myocytes were paced by electrical field stimulation
at 1 Hz by using platinum wires embedded in the walls of the
superfusion chamber throughout all experimental protocols to avoid
changes in cell calcium or shortening due to the chronotropic actions
of ß-receptor agonists. Myocytes were exposed to a bolus of the
nonselective ß-receptor agonist isoproterenol or the
ß2-receptor–selective agonist zinterol by introducing 50
µL of superfusion buffer containing five times the desired final
concentration of drug into the initial prechamber. The fluid rapidly
mixes with the inflow stream and thereby is diluted as it enters the
main 1x1-cm central compartment of the chamber, which is designed to
maintain a constant volume of 250 µL. In preliminary experiments,
we established that vehicle, delivered in an identical fashion, does
not affect the calcium transient or twitch. This protocol provides the
means to rapidly deliver agonist to the myocytes in the experimental
chamber but does not permit measurements under steady state conditions.
However, preliminary experiments indicated that the
isoproterenol-dependent increase in calcium transient and twitch
amplitude were more modest when myocytes were superfused with agonists
for more protracted intervals. This suggests that delivery of drug
rapidly as a bolus minimizes the effects of desensitization mechanisms,
which are simultaneously activated after exposure to agonists.
Measurements were made under control conditions and at peak drug
effect, in general, 90 seconds after the bolus of agonist. Where
indicated, 10-7 mol/L of the ß-receptor subtype
antagonists CGP 20712A (CGP) and ICI 118,551 (ICI) was added to the
superfusion buffer 5 minutes before the bolus of agonist. These highly
selective ß-receptor subtype antagonists by themselves had no
measurable effects on the calcium transient or cell shortening under
these experimental conditions.
Based on the published values for the affinity of zinterol, CGP, and
ICI at ß-adrenergic receptor subtypes,24 25 the
fractional occupancy of ß1- and
ß2-adrenergic receptors by zinterol, in the absence or
presence of 10-7 mol/L CGP or 10-7 mol/L ICI,
was estimated. This was accomplished by using the equation of Neve et
al26 and assuming an equilibrium dissociation constant
(Kd) for zinterol of 1000 nmol/L at
ß1-receptors and 40 nmol/L at ß2-adrenergic
receptors,24 a Kd for CGP of 5
nmol/L at ß1-receptors and 16 000 nmol/L at
ß2-receptors,14 25 and a
Kd for ICI of 1900 nmol/L at
ß1-receptors and 31 nmol/L at
ß2-receptors.25 In the absence of
antagonists, this analysis predicts that 71.4% of the
ß2-receptors and 9.1% of the ß1-receptors
will be occupied by 10-7 mol/L zinterol. In the presence
of 10-7 mol/L ICI, the percentage of
ß2-receptors occupied by zinterol is reduced to 37%,
whereas there is only a trivial reduction in agonist occupancy of
ß1-receptors. In the presence of 10-7 mol/L
CGP, there is no change in agonist occupancy of the
ß2-receptor population, whereas <0.4% of the
ß1-receptors are occupied by zinterol. At a 100-fold
higher concentration of zinterol (10-5 mol/L), 100% of
the ß2-receptors and 90% of the
ß1-receptors are occupied by agonist. In the presence of
10-7 mol/L CGP, occupancy of ß1-receptors by
zinterol is reduced to 28%; agonist occupancy of
ß2-receptors is not affected. Of course, these
calculations assume steady state conditions and do not strictly apply
to our studies of calcium and contractile function, which assessed the
response to agonists delivered as a bolus. Nevertheless, these
calculations provide a useful theoretical estimate of the upper limit
of receptor occupancy by agonists in these studies.
Measurement of cAMP
Intracellular cAMP was measured essentially as described
previously.20 Briefly, neonatal myocytes grown in 22.1-mm
multiwell dishes or 100 000 rod-shaped adult myocytes were
preincubated for 60 minutes at room temperature with 10 mmol/L
theophylline. The phosphodiesterase inhibitor was used in biochemical
experiments for two reasons. First, by increasing cAMP content, it
facilitated measurements of intracellular cAMP. Second, by preventing
cAMP degradation, it permitted us to isolate and assess
agonist-dependent increases in the formation of cAMP. Where indicated,
10-7 mol/L of the ß-receptor subtype antagonists CGP or
ICI were added to the cells 5 minutes before agonist. Assays were
performed for 5 minutes at room temperature and were terminated by
removal of the incubation buffer and addition of 1 mL ethanol. Each
condition was performed on two wells or aliquots of cells (neonatal or
adult myocytes, respectively) and was assayed for cAMP in
quadruplicate. The alcohol-fixed cell extract was boiled for 3 minutes,
cooled, brought to original volume with ethanol, and stored at
-80°C. Aliquots of the supernatant were dried under a stream of
nitrogen, and cAMP in the residue was determined by radioimmunoassay
(New England Nuclear/Du Pont Co).
Radioligand Binding Assay
Cyanopindolol was radioiodinated to a theoretical specific
activity of 2200 Ci/mmol and purified according to methods previously
published.27 Radioligand binding experiments were
performed on broken cell preparations of adult ventricular myocytes and
nonenzymatically resuspended cultured neonatal rat ventricular myocytes
according to methods described previously.28 To
nonenzymatically detach cultured neonatal ventricular myocytes,
monolayers were incubated with cell-detaching solution containing
(mmol/L) NaCl 130, NaHCO3 16, KCl 3,
NaH2PO4 0.5, sucrose 10, and EDTA 1 (EDTA stock
solution adjusted to pH 7.3 with Tris buffer) for 10 minutes at 37°C.
Cells were detached by gentle pipetting, diluted to three times their
volume with similar medium containing 2 mmol/L MgCl2
instead of EDTA, pelleted by centrifugation at 700g for 5
minutes, and then resuspended in the MgCl2-containing
solution at 3x106 cells per milliliter. This
preparation was used to avoid possible enzyme-induced proteolytic
damage to cell-surface receptors and to minimize any loss of receptor
containing material that might accompany the use of more purified
membrane fractions.29 To measure total ß-adrenergic
receptor density and affinity for
[125I]iodocyanopindolol (ICYP), 40 000 neonatal
ventricular myocytes or 20 000 adult ventricular myocytes were
incubated for 1 hour at 37°C with ICYP (5 to 225 pmol/L) in a final
volume of 1 mL. The assay buffer contained 0.15 mol/L NaCl, 0.01 mol/L
KCl, 0.01 mol/L MgCl2, 0.001 mol/L EDTA, 2 mg/mL
dextrose, 1 mg/mL BSA, and 0.01 mol/L Tris, pH 7.4. Specific binding of
ICYP was defined as the component of total binding that could be
inhibited by excess unlabeled propranolol (1 µmol/L) and constituted
90% of total binding at concentrations of ICYP near
Kd. In preliminary experiments, we established
that specific binding of ICYP to neonatal and adult myocyte
preparations reaches equilibrium by 50 minutes, remains stable for at
least an additional 30 minutes, and is linear with cell density up to
60 000 adult and 200 000 neonatal ventricular myocytes per
milliliter. ICYP bound to membrane protein was separated from free,
unbound ICYP by the addition of 1 mL of warmed buffer (37°C) and
rapid vacuum filtration of the entire volume over glass-fiber filters
(Gelman A/E, Gelman Sciences), which was followed by three washes with
5 mL of 10 mmol/L Tris, pH 7.4. Radioactivity trapped by the filters
was detected in a Packard Autogamma scintillation
spectrophotometer. Kd and the maximal number of
binding sites (Bmax) for ICYP were determined by
Scatchard analysis of saturation binding isotherms.
ß2-Adrenergic receptor density in neonatal and adult
ventricular myocytes was measured in parallel in some experiments. The
strategy to assess ß2-adrenergic receptor subtype
expression took advantage of the extraordinarily high degree of
selectivity of CGP for ß1-adrenergic receptors
(Kd, 5 and 16 000 nmol/L at
ß1- and ß2-receptors, respectively, in rat
heart14 25 ). The published values for the affinity of CGP
at ß1- and ß2-adrenergic receptors were
validated for our experimental system in preliminary competition
binding studies (data not shown). Accordingly, myocytes were
preincubated for 5 minutes at 37°C with 5x10-7 mol/L
CGP, and saturation binding experiments were performed precisely as
described above. Because this concentration of CGP selectively binds
and masks ß1-adrenergic receptors, only
ß2-receptors are detected under these experimental
conditions. The percentage of ß2-receptors (measured in
the presence of CGP) relative to total ß-adrenergic receptors
(measured in parallel without CGP) is reported.
Although ß-adrenergic receptors are remarkably stable integral membrane proteins, we also considered the possibility that our approach, which compares ß2-adrenergic receptor responsiveness (modulation of contractile function and stimulation of cAMP accumulation) in intact monolayers and ß2-receptor density in nonenzymatically resuspended myocyte preparations, may not be valid. To determine whether ß2-adrenergic receptors might be selectively lost during the nonenzymatic resuspension protocol, we examined ß-receptor characteristics on intact myocytes in monolayer culture (according to methods previously published by our laboratory27 ). No difference in total ß-receptor density, antagonist affinity, or the proportion of ß1- and ß2-receptors could be detected between neonatal myocytes in monolayer culture or myocytes that had been nonenzymatically resuspended (data not shown). These results argue that the nonenzymatic resuspension does not introduce discernible differences in ß-adrenergic receptor expression.
Statistics
For measurements of intracellular calcium and cell motion, six
successive transients were superimposed and averaged. Amplitude and
width at one-half maximal amplitude were measured for both the calcium
and motion transients. For studies of calcium and contractile function,
all data represent results of experiments on cells from at least
two separate isolations (adult) or cultures (neonate). For cAMP
studies, experiments were performed at least four times on different
myocyte preparations. Data are presented as mean±SEM, and
statistical comparisons were made by using Student's t test
for paired observations or ANOVA for multiple comparisons as indicated.
Significance was defined at the P<.05 level.
Materials
CGP was obtained from CIBA-GEIGY. ICI was purchased from
Cambridge Research Biochemicals Ltd. Zinterol was generously provided
by Bristol-Myers Squibb. Fura 2-AM was purchased from Molecular Probes.
All other chemicals were reagent grade.
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ß-Adrenergic Receptor Subtype Modulation of Calcium and Cell Shortening in Neonatal and Adult Ventricular Myocytes
Our first aim was to compare the ß-adrenergic receptor response of adult and neonatal rat ventricular myocytes. A representative tracing of the cell shortening and calcium transients from an electrically stimulated neonatal ventricular myocyte before and after exposure to a bolus of 10-7 mol/L isoproterenol is illustrated in Fig 1
(top). Typical results from a
comparable experiment performed on a single adult ventricular myocyte
are depicted in Fig 2 (top). In each case, the
nonselective ß-receptor agonist rapidly increased the amplitude of
the calcium transient, and this was accompanied by a marked increase in
the amplitude of cell shortening. Isoproterenol also abbreviated the
duration of the twitch in both neonatal and adult ventricular myocytes.
This is best appreciated in Table 1 and in the lower
right panels of Figs 1 and 2, where signal-averaged calcium and motion
transients in neonatal and adult myocytes before and after exposure to
isoproterenol are superimposed and illustrated on an expanded time
scale.
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It is now well established that the effect of isoproterenol to
accelerate the kinetics of cardiac relaxation is mediated, at least in
part, by the cAMP-dependent phosphorylation of phospholamban, which
results in increased calcium uptake by the sarcoplasmic
reticulum.30 In view of previous biochemical, structural,
and functional data suggesting a more fully developed sarcoplasmic
reticulum in adult than in neonatal myocytes,31 32 we had
anticipated that a developmental difference in sarcoplasmic reticular
function might limit the effect of isoproterenol to enhance relaxation
in myocytes from the newborn ventricle. In this regard, the observation
that isoproterenol markedly accelerates the kinetics of the calcium
transient and cell shortening in neonatal myocytes is noteworthy. In
both neonatal and adult myocytes, the effect of isoproterenol to
increase twitch amplitude was maximal 90 seconds after the bolus of
drug; partial recovery toward the resting value was evident by 200
seconds (Table 1). Although this might be anticipated as a result of a
fall in the concentration of agonist during drug washout, the effect of
the bolus of isoproterenol to abbreviate the duration of the twitch
fully persisted for at least 200 seconds (see Table 1).
The selective ß2-adrenergic agonist zinterol at
10-7 mol/L also modulated the calcium and cell motion
transients of neonatal ventricular myocytes (Fig 3 and
Table 1). Zinterol enhanced the amplitude and hastened the kinetics of
the calcium transient; these changes were accompanied by an increase in
the amplitude and an acceleration in the kinetics of cell shortening.
In contrast, when single adult ventricular myocytes were exposed to
zinterol under identical experimental conditions, a different result
was obtained. As illustrated in the original tracing from a
representative adult ventricular myocyte depicted in Fig 4, a bolus of 10-7 mol/L zinterol had no
effect on the amplitude or the kinetics of the calcium transient or the
twitch.
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Figs 1 through 4 illustrate data from representative experiments
examining the effects of equimolar concentrations of isoproterenol and
zinterol to modulate the calcium and cell motion transients of neonatal
and adult myocytes. Corresponding averaged data that compare the
responses of neonatal and adult myocytes with isoproterenol and
zinterol are presented in Fig 5 and Table 1. In
neonatal myocytes, isoproterenol and zinterol both increase the
amplitude and accelerate the kinetics of the calcium and motion
transients. It is noteworthy that the effect of 10-7 mol/L
zinterol to increase the amplitude of the calcium and motion transients
was only 37.6% and 41.0% as great as the effect of an equimolar
concentration of isoproterenol. In contrast, the effects of equimolar
concentrations of isoproterenol and zinterol to accelerate the kinetics
of the calcium and motion transients were similar in magnitude.
Finally, these results emphasize that under these experimental
conditions zinterol influences the calcium transient and cell
shortening in neonatal, but not in adult, ventricular myocytes.
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Using highly selective ß-adrenergic receptor subtype inhibitors, we
verified that the response to zinterol in neonatal myocytes reflects
the selective activation of ß2-adrenergic receptors (Fig 6). ICI, a selective ß2-adrenergic
receptor antagonist, had no effect on cell shortening alone but
effectively abolished the effect of zinterol to increase the amplitude
and hasten the kinetics of the twitch. In contrast, the response to
zinterol persisted in the presence of CGP, a selective inhibitor of the
ß1-adrenergic receptor.
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Xiao and Lakatta16 recently identified a
ß2-adrenergic receptor response in single adult rat
ventricular myocytes that is qualitatively distinct from the response
to ß1-adrenergic receptor agonists. Specifically,
zinterol was reported to increase the amplitude of the calcium
transient and the twitch without an effect on the time course of the
calcium transient and with only a minor effect to accelerate the
twitch. However, our experiments and those reported by Xiao and Lakatta
use markedly different experimental conditions. In the cited study,
myocytes were superfused for 10 minutes with 10-5 mol/L
zinterol. Indeed, when isolated adult ventricular myocytes were
stimulated with this 100-fold higher concentration of zinterol, either
as a bolus (Fig 7) or according to a superfusion
protocol that mimicked the method described by Xiao and Lakatta (data
not shown), we also consistently observed an increase in the amplitude
of cell shortening. The effect of a bolus of 10-5 mol/L
zinterol to increase the amplitude of the twitch was accompanied by an
increase in the amplitude of the calcium transient; neither the calcium
transient nor the twitch was significantly influenced by a 10-fold
lower concentration of zinterol delivered as a bolus (data not shown).
Moreover, the difference in the characteristics of the responses to
isoproterenol and a high concentration of zinterol, previously noted by
Xiao and Lakatta, was confirmed in our experiments. Whereas
isoproterenol markedly accelerated the kinetics of the twitch, zinterol
did not (Fig 7, open bars).
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Insofar as the stimulatory effects of a high concentration of zinterol
might be anticipated to result from combined drug actions at
ß1- and ß2-adrenergic receptors, we next
determined whether the effect of a bolus of 10-5 mol/L
zinterol to modulate contractile function was influenced by the
ß1-receptor blocker CGP. As illustrated in Fig 7, the
effect of zinterol to increase the amplitude of the twitch was not
significantly attenuated by CGP, but the presence of CGP unmasked an
effect of zinterol to prolong the time course of the twitch (hatched
bars). In the combined presence of CGP and ICI, zinterol did not
significantly alter contractile function (ie, contractile amplitude
declined by 0.3±1.5%, n=3, P=NS). Taken together, these
results constitute evidence that changes in contractile function after
bolus exposure to 10-5 mol/L zinterol result from the
combined activation of ß1- and
ß2-adrenergic receptors.
These experiments confirm the presence of functional
ß2-adrenergic receptors that are linked to an increase in
cell motion in adult rat ventricular myocytes. However, the results
also emphasize two important differences between the characteristics of
the ß2-adrenergic receptor response of neonatal and adult
myocytes, which are illustrated by the representative tracings
presented in Fig 8. First, at 10-7
mol/L zinterol (a concentration that selectively activates
ß2-adrenergic receptors), the response is restricted to
neonatal myocytes, emphasizing that adult myocytes are considerably
more refractory than neonatal myocytes to the stimulatory effects of
ß2-receptor agonists. Second, the effects of
ß2-agonists to abbreviate the calcium transient and
accelerate the kinetics of cell shortening are confined to neonatal
myocytes. Zinterol does not hasten the kinetics of the twitch in adult
ventricular myocytes. In fact, the experiments using the
ß1-receptor subtype–selective blocker CGP to isolate the
ß2-receptor stimulatory effects of zinterol provide
convincing evidence that activation of ß2-adrenergic
receptors results in prolongation of the twitch in adult cardiac
myocytes. Thus, the distinct effect of zinterol on the time course of
contraction in neonatal and adult rat ventricular myocytes reflects a
fundamental age-dependent difference in ß2-receptor
modulation of contractile performance and constitutes the second major
developmental difference in ß2-receptor action.
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ß2-Receptor Activation of cAMP Accumulation
Since ß-receptor agonists are known to stimulate adenylyl
cyclase activity and promote the intracellular accumulation of cAMP, we
next probed for age-dependent differences in the effect of
isoproterenol and zinterol to increase intracellular cAMP. Fig 9 compares the dose-response relation of the effects of
isoproterenol and zinterol on intracellular cAMP accumulation in
neonatal and adult ventricular myocytes. Isoproterenol induced a
dose-dependent increase in intracellular cAMP accumulation, which was
large and of similar magnitude in neonatal and adult ventricular
myocytes (20.0±1.0- and 20.6±2.6-fold over basal in response to
10-5 mol/L isoproterenol, respectively). Zinterol also
increased cAMP accumulation in neonatal and adult myocytes. In neonatal
myocytes, 10-7 mol/L zinterol induced a substantial
increase in cAMP accumulation (7.0±0.4-fold), which represented
38.1% of the response to an equimolar concentration of isoproterenol.
In contrast, zinterol elicited only a very minor increase in cAMP
accumulation in adult ventricular myocytes. Intracellular cAMP levels
were elevated 2.6±0.3-fold over basal in the presence of
10-5 mol/L zinterol, the 100-fold higher agonist
concentration required to modulate contractile function in adult
myocytes.
|
The effect of zinterol to increase cAMP accumulation did not appear to
saturate over a wide range of concentrations, raising the possibility
that zinterol increases cAMP accumulation via an action at a
heterogeneous population of ß-adrenergic receptors. Indeed, up to
10-7 mol/L, the effect of zinterol to increase cAMP
accumulation was completely abolished by ICI but was not attenuated by
CGP, in both neonatal and adult ventricular myocyte preparations (Fig 10). These results provide strong evidence that at
concentrations 
10-7 mol/L, zinterol increases cAMP
accumulation via a highly selective action at
ß2-adrenergic receptors in neonatal and adult ventricular
myocytes. In contrast, at 10-6 mol/L zinterol, a small but
significant increase in cAMP accumulation in the presence of ICI and a
small but significant diminution in cAMP accumulation in the presence
of the highly selective ß1-receptor antagonist CGP were
consistently noted. In separate experiments, we examined the effects of
10-5 mol/L zinterol (the concentration shown to modulate
the calcium transient and the twitch in adult ventricular myocytes).
The effect of 10-5 mol/L zinterol to increase cAMP
accumulation in adult ventricular myocytes was significantly attenuated
by ß1-receptor blockade with CGP (33.2±6.4%) or
ß2-receptor blockade with ICI (45.0±5.1%); it was
virtually abolished when CGP and ICI were simultaneously included in
the assay (87.2±2.2%, P<.05, n=6 for each). These results
indicating that 10-5 mol/L zinterol increases cAMP via
combined actions at ß1- and ß2-adrenergic
receptors are not entirely surprising. Published values for the
relative selectivity of zinterol for ß1- and
ß2-adrenergic receptors (Kd=1000
and 40 nmol/L at ß1- and ß2-adrenergic
receptors, respectively24 ) would predict that the
fractional occupancy of ß1-adrenergic receptors by
zinterol would increase progressively as the concentration of zinterol
rises over this dose range.
|
One could argue that there might be limitations to an approach that compares zinterol responsiveness in myocytes freshly isolated from the adult heart or cultured from the neonatal heart. To determine whether the differences in ß2-receptor actions represent age-dependent differences in ß2-receptor responsiveness rather than changes in the ß2-receptor signaling pathway (which are induced by cell culture), we assessed ß2-receptor responsiveness in freshly isolated neonatal myocytes. Since the myocytes freshly isolated from the neonatal heart are rounded and not amenable to studies of agonist-induced changes in contractility, the effect of zinterol to increase cAMP was used as a measure of ß2-receptor responsiveness in these experiments. When compared with the response to an equimolar concentration of isoproterenol, zinterol induced an equivalent increase in cAMP accumulation in acutely isolated neonatal ventricular myocytes and myocytes cultured for 5 days (33.3±8.3% and 41.7±8.3%, respectively; n=4, P=NS). These results argue that the culture procedure itself does not introduce differences in ß2-receptor responsiveness.
ß-Receptor Subtype Expression in Neonatal and Adult Rat
Ventricular Myocytes
Buxton and Brunton33 previously reported that within
the limits of detection using radioligand binding with ICYP, purified
adult rat ventricular myocytes possess a homogeneous population of
ß1-adrenergic receptors. However, the observation that a
low concentration of a ß2-adrenergic receptor agonist
preferentially modulates the calcium and cell-shortening transients and
increases cAMP accumulation in neonatal myocytes raised the possibility
that neonatal myocytes might be relatively enriched in
ß2-adrenergic receptors. Therefore, we compared
ß-adrenergic receptor characteristics in ventricular myocytes from
the neonatal and adult heart (Table 2). In each case,
ICYP identified a single class of high-affinity ß-adrenergic
receptors. Scatchard analysis of the linear saturation isotherms
revealed a higher total ß-receptor density in adult myocytes than in
neonatal myocytes, when the value was expressed as sites per cell.
However, we reasoned that a comparison of ß-receptor expression in
neonatal and adult myocytes must take into account the marked
difference in size and surface area between these cells. By measuring
the diameter of the spherical resuspended neonatal ventricular myocytes
(11.5±0.4 µm, n=60), we estimated that the cell-surface density of
ß-adrenergic receptors in neonatal ventricular myocytes is 107
sites per square micrometer. In comparison, assuming that adult
ventricular myocytes are brick-shaped (with dimensions of 127.0±4.5
µm in length, 25.1±1.7 µm in width, and 10 µm in depth;
n=10), we estimated a fivefold lower cell surface ß-adrenergic
receptor density in adult myocytes (20 sites per square micrometer).
These results in adult ventricular myocytes are in reasonable accord
with the calculations reported previously by Buxton and Brunton. We
recognize that this rather simple approach to correct Bmax
for age-dependent differences in cell size does not take into account
potential membrane infolding, which may be significant and may differ
between neonatal and adult myocytes. However, the accuracy of other
approaches to calculate membrane surface area also is uncertain, since
receptor distribution may not be uniform within specialized membrane
domains. Thus, the present analysis suggests that cell surface
ß-adrenergic receptor density may be higher in neonatal than in adult
ventricular myocytes.
|
To determine the relative proportion of ß1- and
ß2-receptors in ventricular myocytes, saturation binding
experiments were performed in parallel in the presence of
5x10-7 mol/L CGP (Table 2). Given the >1000-fold
selectivity of CGP for ß1-adrenergic
receptors,14 25 this concentration of CGP completely and
specifically masks the ß1-adrenergic receptor population;
only ß2-adrenergic receptors are available to be
identified by the radioligand under these experimental conditions.
Using this strategy, we demonstrated that the ß2-receptor
population constitutes a similar minor proportion of the total
ß-receptor population in neonatal and adult ventricular myocytes
(16.7±2.3% and 16.9±0.9% of the total ß-receptor density in the
corresponding experimental preparation, respectively; n=3 for neonate
and n=4 for adult; P=NS).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
These studies are the first to demonstrate that ß2-adrenergic receptor agonists modulate calcium homeostasis and contractile performance in neonatal rat ventricular myocytes. In fact, the data reported herein argue that ß2-adrenergic receptors play an even more important role in mediating the contractile response to catecholamines in the neonatal than in the adult rat heart. This conclusion is based on the finding that a low concentration of ß2-adrenergic receptor agonists, which is ineffective in adult ventricular myocytes, rapidly and profoundly modulates the calcium transient and cell contraction in neonatal ventricular myocytes. The notion that the ß2-receptor response may play an important role in mediating the contractile response to catecholamines in the neonatal heart is particularly intriguing when considered in the context of normal cardiac development. Since norepinephrine has at least a 10-fold higher affinity for ß1- than for ß2-adrenergic receptors, several investigators have speculated that ß1-adrenergic receptors preferentially mediate the response to neuronally released norepinephrine. On the other hand, it has been postulated that ß2-adrenergic receptors mediate the response to circulating epinephrine.3 34 35 In this context, it is important to note that maturation of sympathetic innervation is not complete in the neonatal rat heart. Thus, it becomes tempting to speculate that the exaggerated ß2-receptor response in the noninnervated neonatal heart might provide vital support of contractile function. The logical extension of this hypothesis is that with maturation of sympathetic innervation, the ß2-receptor response becomes less critical and is lost. Indeed, sympathetic innervation of the heart has been shown to play an important role in the maturation of several aspects of cardiac cell function. In vitro innervation of previously noninnervated neonatal rat ventricular myocytes has been shown to increase the expression of functional L-type calcium channels,36 alter the gating of the voltage-gated sodium channel,18 modulate contractile function,37 and alter
1-adrenergic receptor
responsiveness.38 Although it has been speculated that
neurally released norepinephrine might regulate
ß1-receptor expression,25 currently there is
no compelling evidence that norepinephrine or other trophic factors
released by sympathetic neurons regulate ß2-adrenergic
receptor expression or function. Whether sympathetic innervation acts
as a potential modulator of ß2-receptor responsiveness
remains to be elucidated. These studies also provide evidence that ß2-receptors act, at least in part, via distinct intracellular signaling mechanisms to modulate contractile function in neonatal and adult ventricular myocytes. There are two major differences in the characteristics of the response to ß2-adrenergic receptor stimulation between neonatal and adult myocytes that support this conclusion. First, the effect of zinterol to accelerate the kinetics of the calcium transient and cell shortening is restricted to neonatal myocytes. This effect was not confined to low concentrations; qualitatively similar responses were observed when neonatal myocytes were exposed to higher concentrations of zinterol (data not shown). In contrast, experiments using CGP to block any ß1-receptor–stimulatory actions of zinterol provide evidence that ß2-receptor stimulation leads to a prolongation of the kinetics of the twitch in adult myocytes. It is important to note that the absence of an effect of zinterol to accelerate the time course of the twitch in adult myocytes is not simply a result of the very modest ß2-receptor agonist–dependent increase in intracellular cAMP in these cells. This conclusion is based on data from control experiments demonstrating that low concentrations of isoproterenol (10-9 to 10-8 mol/L), estimated to elevate intracellular cAMP to a level equivalent to that achieved in the presence of 10-5 mol/L zinterol, effectively accelerated the kinetics of the twitch (data not shown). Thus, these results argue for the presence of a fundamental age-dependent difference in ß2-receptor modulation of the kinetics of contraction. This result is noteworthy in light of the previous apparently contradictory findings that ß2-adrenergic receptor agonists prolong the time course of contraction in feline papillary muscle preparations,39 induce at most only minor effects to abbreviate the twitch in adult rat ventricular myocytes,16 but accelerate the kinetics of contraction in strips of human atrial myocardium.40 Prior studies have attempted to attribute some of these inconsistencies to species-dependent differences in ß2-receptor action. Our results provide compelling evidence that developmental changes in the characteristics of the ß2-receptor response also must be considered.
The second fundamental difference between the characteristics of the response to ß2-adrenergic receptor agonists between neonatal and adult myocytes relates to stimulation of cAMP accumulation. In neonatal myocytes, there was a close correlation between the effect of zinterol to promote intracellular cAMP accumulation, to increase the amplitude of the calcium transient, and to augment cell shortening. This is consistent with the notion that ß2-receptor agonists modulate contractile performance primarily via a cAMP-dependent mechanism in the neonatal heart. Nevertheless, future studies to assess whether there is a concordance between the agonist-dependent activation of protein kinase A and modulation of contractile function will be required to substantiate this conclusion. In contrast, the effect of zinterol to increase the amplitude of the twitch and the calcium transient was associated with only a modest increase in intracellular cAMP accumulation in adult myocytes. Although it is widely accepted that ß-receptor agonists modulate cardiac contractile function through an action to stimulate adenylyl cyclase and increase intracellular cAMP, discrepancies between agonist-dependent elevations in cellular cAMP content and increases in inotropy had been noted previously.41 There are several explanations that can be proposed to reconcile these discrepancies. For example, evidence that receptor-mediated increases in inotropy correlate best with receptor-dependent increases in cAMP in the particulate fraction of the cell41 42 raise the possibility that ß2-adrenergic receptors might modulate contractile function by increasing a specific pool of cAMP and/or cAMP-dependent protein kinase. Alternatively, it could be argued that ß2-adrenergic receptors increase inotropy without enhancing relaxation by stimulating a cAMP-independent membrane-delimited process. Direct Gs-dependent activation of voltage-sensitive calcium channels could constitute such a candidate mechanism.43 In this regard, recent evidence that ß1- and ß2-receptor agonists induce an equivalent increase in peak calcium current amplitude but that ß2-receptor stimulation preferentially prolongs calcium current inactivation is noteworthy.16 Finally, results of a previous study suggest that for a given increase in twitch amplitude, the increase in the amplitude of the calcium transient is much more modest for ß2- than for ß1-receptor agonists.16 Of note, this conclusion is based on experiments that compare the effect of zinterol with the effect of norepinephrine (each superfused for 10 minutes); the difference between ß-receptor subtype actions to increase the amplitude of the calcium transient and the twitch was less pronounced in the present study, which compares zinterol with the mixed ß-receptor agonist isoproterenol under conditions in which isoproterenol rapidly and transiently activates the ß-adrenergic receptor. Nevertheless, this observation has raised the possibility that ß1- and ß2-receptor agonists influence myofilament responsiveness to calcium in a distinct fashion. The recent demonstration that the ß2-adrenergic receptor activates Na-H exchange via a G protein other than Gs and a mechanism that is independent of cAMP44 may be relevant, since alkalinization resulting from receptor-dependent stimulation of Na-H exchange would be predicted to increase the calcium sensitivity of the contractile proteins.45 Future studies to explore whether ß2-receptor agonist activation of Na-H exchange accounts for the ß2-receptor–dependent increase in the amplitude of the twitch with no increase or only a minor increase in intracellular calcium will be of interest.
Results reported herein and a previous study by Xiao and Lakatta16 provide unambiguous evidence for ß2-receptor modulation of contractile function in adult rat ventricular myocytes. However, a difference between the two studies deserves comment. In the previous study, the effect of superfusion with 10-5 mol/L zinterol to increase the amplitude without significantly accelerating the kinetics of the twitch in adult ventricular myocytes was attenuated by ICI but remained completely intact in the presence of CGP. This observation was taken as strong evidence that superfusion with a high concentration of zinterol exclusively activates the ß2-receptor population. In the studies reported herein, the effect of 10-5 mol/L zinterol to increase intracellular cAMP accumulation was dependent on the combined actions at ß1- and ß2-adrenergic receptors. Similarly, the effect of 10-5 mol/L zinterol, delivered rapidly as a bolus, to modulate contractile function was influenced by CGP. These observations constitute strong evidence that both ß1- and ß2-adrenergic receptor populations contribute to the functional response to zinterol under our experimental conditions. The precise protocol for drug delivery (rapid bolus versus prolonged continuous superfusion) constitutes the most striking difference between the experimental design used to investigate agonist modulation of contractile function in these two studies. Whether ß-receptor subtypes differ in their kinetics of desensitization or regulation in the cell, thereby accounting for the discrepancy in the ß-receptor subtype selectivity of 10-5 mol/L zinterol noted in these studies, will require further study.
It has recently become evident that several important structural and regulatory proteins in the heart undergo maturational and regionally regulated expression. In some cases, disease-associated changes in protein expression that recapitulate the ontogenic changes and may provide an adaptive response to the pathological state have been identified.46 47 With regard to the ß-adrenergic receptor, it is increasingly evident that various pathological states are associated with dramatic changes in ß-adrenergic receptor expression and function in the heart.1 This has been studied most intensively in the setting of heart failure, where a diminished cAMP and inotropic response to ß-receptor agonists, in general, has been associated with a decrease in the density of cell surface ß-adrenergic receptors.3 4 Interestingly, a selective downregulation of the ß1-adrenergic receptor population in the face of a relatively constant density of ß2-adrenergic receptors, which results in a relative increase in the proportion of ß2-adrenergic receptors in the failing heart, has been reported.3 4 48 The relative increased number of ß2-adrenergic receptors retains almost full inotropic activity, thereby helping to support contractile function in the failing heart. In this context, results reported herein that establish an age-dependent difference in ß-receptor subtype function and suggest an age-dependent difference in the density of cell surface ß-adrenergic receptors, without any change in the proportion of ß2-receptors, are intriguing. Studies to further characterize ß2-receptor coupling to intracellular signaling mechanisms in the context of cardiac development are in progress. However, in the context of the known disease-associated changes in ß-receptor subtype expression and function, results reported herein may suggest that adaptive changes in ß-receptor subtype function in heart failure constitute, at least in part, yet another instance of a disease-associated recapitulation of the neonatal phenotype.
In summary, the present study demonstrates that low concentrations of ß2-receptor agonists stimulate cAMP accumulation and modulate contractile function in neonatal but not adult ventricular myocytes. Although at high concentrations zinterol also modulates contractile function in adult myocytes, the distinct characteristics of the biochemical and contractile responses to zinterol in neonatal and adult myocytes provide persuasive evidence that age-dependent differences in ß2-receptor coupling to more distal elements in the signaling cascade can influence the nature of the ß2-adrenergic response.
|
Acknowledgments |
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This study was supported by US Public Health Service–National Heart, Lung, and Blood Institute grant HL-28958.
Received April 20, 1994; accepted September 30, 1994.
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F. Rochais, A. Abi-Gerges, K. Horner, F. Lefebvre, D. M.F. Cooper, M. Conti, R. Fischmeister, and G. Vandecasteele A Specific Pattern of Phosphodiesterases Controls the cAMP Signals Generated by Different Gs-Coupled Receptors in Adult Rat Ventricular Myocytes Circ. Res., April 28, 2006; 98(8): 1081 - 1088. [Abstract] [Full Text] [PDF]
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L. Barki-Harrington, C. Perrino, and H. A Rockman Network integration of the adrenergic system in cardiac hypertrophy Cardiovasc Res, August 15, 2004; 63(3): 391 - 402. [Abstract] [Full Text] [PDF]
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A. Besana, A. Barbuti, M. A. Tateyama, A. J. Symes, R. B. Robinson, and S. J. Feinmark Activation of Protein Kinase C {epsilon} Inhibits the Two-pore Domain K+ Channel, TASK-1, Inducing Repolarization Abnormalities in Cardiac Ventricular Myocytes J. Biol. Chem., August 6, 2004; 279(32): 33154 - 33160. [Abstract] [Full Text] [PDF]
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A. Arutunyan, A. Pumir, V. Krinsky, L. Swift, and N. Sarvazyan Behavior of ectopic surface: effects of {beta}-adrenergic stimulation and uncoupling Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2531 - H2542. [Abstract] [Full Text] [PDF]
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J. J. Saucerman, L. L. Brunton, A. P. Michailova, and A. D. McCulloch Modeling {beta}-Adrenergic Control of Cardiac Myocyte Contractility in Silico J. Biol. Chem., November 28, 2003; 278(48): 47997 - 48003. [Abstract] [Full Text] [PDF]
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M. J. Lohse, S. Engelhardt, and T. Eschenhagen What Is the Role of {beta}-Adrenergic Signaling in Heart Failure? Circ. Res., November 14, 2003; 93(10): 896 - 906. [Abstract] [Full Text] [PDF]
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J.-A. Yao, D. E. Gutstein, F. Liu, G. I. Fishman, and A. L. Wit Cell Coupling Between Ventricular Myocyte Pairs From Connexin43-Deficient Murine Hearts Circ. Res., October 17, 2003; 93(8): 736 - 743. [Abstract] [Full Text] [PDF]
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V. O. Rybin, P. W. Grabham, H. Elouardighi, and S. F. Steinberg Caveolae-associated proteins in cardiomyocytes: caveolin-2 expression and interactions with caveolin-3 Am J Physiol Heart Circ Physiol, June 5, 2003; 285(1): H325 - H332. [Abstract] [Full Text] [PDF]
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V. O. Rybin, A. Sabri, J. Short, J. C. Braz, J. D. Molkentin, and S. F. Steinberg Cross-regulation of Novel Protein Kinase C (PKC) Isoform Function in Cardiomyocytes. ROLE OF PKCepsilon IN ACTIVATION LOOP PHOSPHORYLATIONS AND PKCdelta IN HYDROPHOBIC MOTIF PHOSPHORYLATIONS J. Biol. Chem., April 11, 2003; 278(16): 14555 - 14564. [Abstract] [Full Text] [PDF]
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S. Bartel, E.-G. Krause, G. Wallukat, and P. Karczewski New insights into {beta}2-adrenoceptor signaling in the adult rat heart Cardiovasc Res, March 1, 2003; 57(3): 694 - 703. [Abstract] [Full Text] [PDF]
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A. Barbuti, S. Ishii, T. Shimizu, R. B. Robinson, and S. J. Feinmark Block of the background K+ channel TASK-1 contributes to arrhythmogenic effects of platelet-activating factor Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2024 - H2030. [Abstract] [Full Text] [PDF]
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R. S. Ostrom, C. Gregorian, R. M. Drenan, Y. Xiang, J. W. Regan, and P. A. Insel Receptor Number and Caveolar Co-localization Determine Receptor Coupling Efficiency to Adenylyl Cyclase J. Biol. Chem., November 2, 2001; 276(45): 42063 - 42069. [Abstract] [Full Text] [PDF]
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S. Wei, E. C. Rothstein, L. Fliegel, L. J. Dell'Italia, and P. A. Lucchesi Differential MAP kinase activation and Na+/H+ exchanger phosphorylation by H2O2 in rat cardiac myocytes Am J Physiol Cell Physiol, November 1, 2001; 281(5): C1542 - C1550. [Abstract] [Full Text] [PDF]
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E. Devic, Y. Xiang, D. Gould, and B. Kobilka beta -Adrenergic Receptor Subtype-Specific Signaling in Cardiac Myocytes from beta 1 and beta 2 Adrenoceptor Knockout Mice Mol. Pharmacol., September 1, 2001; 60(3): 577 - 583. [Abstract] [Full Text] [PDF]
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J. Qu, A. Barbuti, L. Protas, B. Santoro, I. S. Cohen, and R. B. Robinson HCN2 Overexpression in Newborn and Adult Ventricular Myocytes : Distinct Effects on Gating and Excitability Circ. Res., July 6, 2001; 89 (1): e8 - e14. [Abstract] [Full Text] [PDF]
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A. Chesley, M. S. Lundberg, T. Asai, R.-P. Xiao, S. Ohtani, E. G. Lakatta, and M. T. Crow The {beta}2-Adrenergic Receptor Delivers an Antiapoptotic Signal to Cardiac Myocytes Through Gi-Dependent Coupling to Phosphatidylinositol 3'-Kinase Circ. Res., December 8, 2000; 87(12): 1172 - 1179. [Abstract] [Full Text] [PDF]
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E. A. Sosunov, R. Z. Gainullin, N.S. Moise, S. F. Steinberg, P. Danilo Jr., and M. R. Rosen {beta}1 and {beta}2-adrenergic receptor subtype effects in German shepherd dogs with inherited lethal ventricular arrhythmias Cardiovasc Res, November 1, 2000; 48(2): 211 - 219. [Abstract] [Full Text] [PDF]
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R.-P. Xiao Cell Logic for Dual Coupling of a Single Class of Receptors to Gs and Gi Proteins Circ. Res., October 13, 2000; 87(8): 635 - 637. [Full Text] [PDF]
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Z. Nagykaldi, D. Kem, R. Lazzara, and B. Szabo Conditioning of beta 1-adrenoceptor effect via beta 2-subtype on L-type Ca2+ current in canine ventricular myocytes Am J Physiol Heart Circ Physiol, September 1, 2000; 279(3): H1329 - H1337. [Abstract] [Full Text] [PDF]
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H. K. Ranu, J. C. W. Mak, P. J. Barnes, and S. E. Harding Gi-dependent suppression of beta 1-adrenoceptor effects in ventricular myocytes from NE-treated guinea pigs Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H1807 - H1814. [Abstract] [Full Text] [PDF]
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A. Sabri, E. Pak, S. A. Alcott, B. A. Wilson, and S. F. Steinberg Coupling Function of Endogenous {alpha}1- and {beta}-Adrenergic Receptors in Mouse Cardiomyocytes Circ. Res., May 26, 2000; 86(10): 1047 - 1053. [Abstract] [Full Text] [PDF]
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J. G. Meszaros, A. M. Gonzalez, Y. Endo-Mochizuki, S. Villegas, F. Villarreal, and L. L. Brunton Identification of G protein-coupled signaling pathways in cardiac fibroblasts: cross talk between Gq and Gs Am J Physiol Cell Physiol, January 1, 2000; 278(1): C154 - C162. [Abstract] [Full Text] [PDF]
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C. Communal, K. Singh, D. B. Sawyer, and W. S. Colucci Opposing Effects of {beta}1- and {beta}2-Adrenergic Receptors on Cardiac Myocyte Apoptosis : Role of a Pertussis Toxin-Sensitive G Protein Circulation, November 30, 1999; 100(22): 2210 - 2212. [Abstract] [Full Text] [PDF]
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R.-P. Xiao, H. Cheng, Y.-Y. Zhou, M. Kuschel, and E. G. Lakatta Recent Advances in Cardiac {beta}2-Adrenergic Signal Transduction Circ. Res., November 26, 1999; 85(11): 1092 - 1100. [Abstract] [Full Text] [PDF]
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S. F. Steinberg The Molecular Basis for Distinct {beta}-Adrenergic Receptor Subtype Actions in Cardiomyocytes Circ. Res., November 26, 1999; 85(11): 1101 - 1111. [Full Text] [PDF]
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Y.-Y. Zhou, H. Cheng, L.-S. Song, D. Wang, E. G. Lakatta, and R.-P. Xiao Spontaneous beta 2-Adrenergic Signaling Fails To Modulate L-Type Ca2+ Current in Mouse Ventricular Myocytes Mol. Pharmacol., September 1, 1999; 56(3): 485 - 493. [Abstract] [Full Text]
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A. M. Feldman and C. McTiernan New Insight Into the Role of Enhanced Adrenergic Receptor-Effector Coupling in the Heart Circulation, August 10, 1999; 100(6): 579 - 582. [Full Text] [PDF]
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F. Schroder and S. Herzig Effects of beta 2-adrenergic stimulation on single-channel gating of rat cardiac L-type Ca2+ channels Am J Physiol Heart Circ Physiol, March 1, 1999; 276(3): H834 - H843. [Abstract] [Full Text] [PDF]
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R.-P. Xiao, P. Avdonin, Y.-Y. Zhou, H. Cheng, S. A. Akhter, T. Eschenhagen, R. J. Lefkowitz, W. J. Koch, and E. G. Lakatta Coupling of ß2-Adrenoceptor to Gi Proteins and Its Physiological Relevance in Murine Cardiac Myocytes Circ. Res., January 22, 1999; 84(1): 43 - 52. [Abstract] [Full Text] [PDF]
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M. A. Laflamme and P. L. Becker Do beta 2-adrenergic receptors modulate Ca2+ in adult rat ventricular myocytes? Am J Physiol Heart Circ Physiol, April 1, 1998; 274(4): H1308 - H1314. [Abstract] [Full Text] [PDF]
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D. R. Wagner, A. Combes, C. McTiernan, V. J. Sanders, B. Lemster, and A. M. Feldman Adenosine Inhibits Lipopolysaccharide-Induced Cardiac Expression of Tumor Necrosis Factor-{alpha} Circ. Res., January 23, 1998; 82(1): 47 - 56. [Abstract] [Full Text] [PDF]
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K. Wong, K. R Boheler, J. Bishop, M. Petrou, and M. H Yacoub Clenbuterol induces cardiac hypertrophy with normal functional, morphological and molecular features Cardiovasc Res, January 1, 1998; 37(1): 115 - 122. [Abstract] [Full Text] [PDF]
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V. A. Skeberdis and a. R. Fischmeister Beta-2 Adrenergic Activation of L-Type Ca++ Current in Cardiac Myocytes J. Pharmacol. Exp. Ther., November 1, 1997; 283(2): 452 - 461. [Abstract] [Full Text]
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L. C. Hool and R. D. Harvey Role of beta 1- and beta 2-adrenergic receptors in regulation of Cl- and Ca2+ channels in guinea pig ventricular myocytes Am J Physiol Heart Circ Physiol, October 1, 1997; 273(4): H1669 - H1676. [Abstract] [Full Text] [PDF]
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J. J. Smolich, H. S. Cox, P. J. Berger, A. M. Walker, G. Eisenhofer, and M. D. Esler Left Ventricular Norepinephrine and Epinephrine Kinetics at Birth in Lambs Circ. Res., September 19, 1997; 81(3): 438 - 447. [Abstract] [Full Text]
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G. E. Billman, L. C. Castillo, J. Hensley, C. M. Hohl, and R. A. Altschuld ß2-Adrenergic Receptor Antagonists Protect Against Ventricular Fibrillation : In Vivo and In Vitro Evidence for Enhanced Sensitivity to ß2-Adrenergic Stimulation in Animals Susceptible to Sudden Death Circulation, September 16, 1997; 96(6): 1914 - 1922. [Abstract] [Full Text]
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L. G. Meggs, H.-e. Huang, B. Li, P. Li, J. Coupet, C. V. Hamby, M. Akanuma, Y. Ishikawa, and P. Anversa Coronary artery stenosis in rats affects {beta}-adrenergic receptor signaling in myocytes Cardiovasc Res, January 1, 1997; 33(1): 98 - 109. [Abstract] [Full Text] [PDF]
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H.-T. Li, N. Y. Honbo, and J. S. Karliner Chronic Hypoxia Increases ß1-Adrenergic Receptor mRNA and Density but Not Signaling in Neonatal Rat Cardiac Myocytes Circulation, December 15, 1996; 94(12): 3303 - 3310. [Abstract] [Full Text]
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V. Rybin and S. F. Steinberg Thyroid Hormone Represses Protein Kinase C Isoform Expression and Activity in Rat Cardiac Myocytes Circ. Res., September 1, 1996; 79(3): 388 - 398. [Abstract] [Full Text]
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T. Jiang, E. Pak, H. Zhang, R. P. Kline, and S. F. Steinberg Endothelin-Dependent Actions in Cultured AT-1 Cardiac Myocytes : The Role of the {varepsilon} Isoform of Protein Kinase C Circ. Res., April 1, 1996; 78(4): 724 - 736. [Abstract] [Full Text]
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D. Keenan, A. Romani, and A. Scarpa Differential Regulation of Circulating Mg2+ in the Rat by ß1- and ß2-Adrenergic Receptor Stimulation Circ. Res., November 1, 1995; 77(5): 973 - 983. [Abstract] [Full Text]
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S. F. Steinberg, S. Alcott, E. Pak, D. Hu, L. Protas, N. S. Moise, R. B. Robinson, and M. R. Rosen beta 1-Receptors increase cAMP and induce abnormal Cai cycling in the German shepherd sudden death model Am J Physiol Heart Circ Physiol, April 1, 2002; 282(4): H1181 - H1188. [Abstract] [Full Text] [PDF]
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A. Barbuti, S. Ishii, T. Shimizu, R. B. Robinson, and S. J. Feinmark Block of the background K+ channel TASK-1 contributes to arrhythmogenic effects of platelet-activating factor Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2024 - H2030. [Abstract] [Full Text] [PDF]
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