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CARDIOVASCULAR
Cardiovascular Research Laboratories, Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada
Received January 30, 2003; accepted March 27, 2003.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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-adrenergic receptors have profound effects on
contraction (Reuter, 1974
),
mediated by phosphorylation of various protein targets
(Movsesian, 1999).
Furthermore, defects in phosphorylation of proteins involved in initiation of
contraction may contribute substantially to contractile dysfunction in heart
failure (Sjaastad et al.,
2003).
Elevation of cAMP activates protein kinase A and promotes phosphorylation
of a number of protein targets important in regulation of strength of
contraction, including L-type Ca2+ channels, phospholamban,
ryanodine receptors, and troponin I
(Movsesian, 1999).
Phosphorylation of L-type Ca2+ channels increases the magnitude of
L-type Ca2+ current (ICa-L)
(McDonald et al., 1994), which
may increase contraction by increasing sarcoplasmic reticulum (SR) stores of
Ca2+ available for release. Also, increased ICa-L may
provide a stronger trigger for SR Ca2+ release through a process
called Ca2+-induced Ca2+ release (CICR)
(Fabiato, 1983).
In CICR a small influx of Ca2+ triggers a large release of
Ca2+ from the SR. The influx of Ca2+ that triggers CICR
in heart is believed to enter the cell primarily as ICa-L.
Contractions and Ca2+ transients initiated by CICR
characteristically are proportional to the amplitude of ICa-L
(London and Krueger, 1986;
Barcenas-Ruiz and Wier, 1987;
Beuckelmann and Wier, 1988;
duBell and Houser, 1989;
Cleemann and Morad, 1991).
Thus, ICa-L not only triggers CICR but also grades the release of
SR Ca2+ so that contractions and transients are large when the peak
amplitude of ICa-L is large, and small when the magnitude of
ICa-L is small. Therefore, one would predict that agents that
increase ICa-L would increase the amount of Ca2+
released from the SR.
Although Ca2+ release is believed to be proportional to
ICa-L, several studies have reported that this proportionality is
lost under specific experimental conditions
(Hussain and Orchard, 1997;
Mackiewicz et al., 2000;
Emanuel et al., 2001;
Ferrier and Howlett, 2001;
Viatchenko-Karpinski and Gyorke,
2001). For example, in cells exposed to the nonselective
-adrenergic agonist isoproterenol, contraction-voltage relations are
sigmoidal, although current-voltage (I-V) relations for ICa-L
remain bell-shaped (Hussain and Orchard,
1997; Viatchenko-Karpinski and
Gyorke, 2001). It is likely that this effect is mediated through
stimulation of cAMP production, because ventricular myocytes dialyzed with
intracellular patch pipette solutions containing 8-bromo-cAMP at 37°C also
exhibit sigmoidal contraction-voltage relationships
(Ferrier et al., 1998;
Mackiewicz et al., 2000). One
explanation that has been advanced to explain this deviation from
proportionality to ICa-L is that elevation of intracellular cAMP
sensitizes CICR so that even very small Ca2+ currents trigger large
contractions and Ca2+ transients
(Hussain and Orchard, 1997;
Wier and Balke, 1999;
Piacentino et al., 2000;
Viatchenko-Karpinski and Gyorke,
2001). Thus, it is possible that stimulatory agents such as
-agonists might alter the proportionality between amplitude of
ICa-L and magnitude of SR Ca2+ release so that small
currents elicit large responses.
Sigmoidal contraction-voltage relations have been reported in several
studies that included 8-bromo-cAMP in patch pipette solutions
(Ferrier et al., 1998;
Mackiewicz et al., 2000).
However, another study that used Tris-cAMP, reported broadening of the
conventional relation, although the curve remained bell-shaped
(Piacentino et al., 2000).
These observations suggest that these two analogs of cAMP are not equally
effective in altering the sensitivity of CICR. We hypothesize that this
disparity might reflect a difference in degradation of these analogs, because
8-bromo-cAMP is resistant to degradation by phosphodiesterase (PDE), whereas
Tris-cAMP is not (Meyer and Miller,
1974). Therefore, the objectives of this study were to compare the
effects of different PDE-resistant and PDE-sensitive analogs of cAMP on CICR
and to determine whether elevation of intracellular cAMP alters CICR so that
small currents elicit large Ca2+ transients and contractions.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). Myocytes
were placed in an experimental chamber on the stage of an inverted microscope
(Eclipse TE200; Nikon, Tokyo, Japan) and superfused at 3 ml/min with a
solution at 37°C containing 45 mM NaCl, 100 mM choline Cl, 2 mM
CaCl2, 4 mM KCl, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES
(pH 7.4 with NaOH), and 300 µM lidocaine to block Na+ current.
In some experiments, switches either to nominally Ca2+-free or
Cd2+-containing solutions at 37°C were made within 300 ms by a
computer-controlled rapid solution changer
(Ferrier et al., 2000).
Voltage Clamp. Discontinuous single electrode voltage-clamp
recordings (sample rate 7–8 kHz) were made with an Axoclamp 2A
voltage-clamp amplifier (Axon Instruments, Inc., Foster City, CA). Experiments
were conducted with 1 to 3 M
patch pipettes filled with
Na+-free solution to inhibit Ca2+ influx by way of
Na+-Ca2+ exchange. The composition of this solution was
70 mM KCl, 70 mM K aspartate, 4 mM MgATP, 1 mM MgCl2, 2.5 mM
KH2PO4, 0.12 mM CaCl2, 0.5 mM EGTA, and 10 mM
HEPES, pH 7.2, with KOH. The pipette solutions were calculated to contain
approximately 46 nM free Ca2+ (Ecal for Windows version 1.1, 1996;
Biosoft, Ferguson, MO). Pipette-to-bath liquid junction potentials were
measured (–8 to –10 mV) and compensated before data acquisition.
An agar bridge (2.7 M KCl) was used as a bath ground to minimize changes in
bath-to-ground liquid junction potentials with solution changes.
cAMP Analogs. Various PDE-resistant and PDE-sensitive analogs of cAMP were added to the patch pipette solutions for specific protocols. The following procedures were followed rigorously to minimize degradation of cAMP. Stock solutions of 0.1 mM cAMP were prepared in deionized water. Stock cAMP was added to aliquots of intracellular solution to give a final concentration of 50 µM cAMP. The aliquots of intracellular solution were frozen at –70°C for no longer than 2 weeks. Aliquots of intracellular solution were thawed on the day of use. cAMP containing solutions were never subjected to more than one cycle of freezing and thawing. Once thawed, intracellular solutions were kept on ice throughout experiments. Pipettes were filled with intracellular solutions by way of plastic syringes that also were kept on ice.
Measurements and Analysis. Unloaded cell shortening was recorded at 120 Hz with a television camera and video edge detector (Crescent Electronics, Sandy, Utah). Ca2+ transients were detected with a DeltaRAM system (Photon Technology International, Brunswick, NJ). Myocytes were loaded with 2.0 µM Fura-2/acetoxymethyl ester (Fura-2/AM) for 20 min in the dark at room temperature. After loading, extracellular dye was washed from the myocytes with extracellular buffer solution for 20 min. Fluorescence signals were collected from a field slightly smaller than the size of a single myocyte. The field was adjusted for each cell with an adjustable aperture. Dye was excited at wavelengths of 340 and 380 nm, and fluorescence emission ratio was measured at 510 nm. Background fluorescence was subtracted before ratios were determined. Recording times were kept as brief as possible to minimize washout and photobleaching of fura-2.
Currents and contractions were acquired and analyzed with pClamp (version 6.0 or 8.0). Recordings were digitized with either a Labmaster TL-125 or a Digidata 1322A A/D interface (Axon Instruments, Inc.) at sample rates up to 50 kHz and stored on a computer for later analysis. Emission ratios were acquired at 100 Hz and analyzed with Felix software (Photon Technology International). Emission ratios were converted to Ca2+ concentrations with a calibration curve determined in vitro with the same optical path used for experiments. Voltage-clamp test steps were preceded by trains of 10 conditioning pulses at 2 Hz, to provide a consistent activation history. Conditioning pulses were 200-ms-long pulses to 0 mV from the holding potential of –80 mV. Test steps were made from a postconditioning potential that followed the conditioning pulse train. Additional details of voltage-clamp protocols are provided in specific results sections.
Peak inward ICa-L was measured as the difference between the maximum inward deflection and a reference point 200 ms after the beginning of the step. When Ca2+ tail currents were measured, ICa-L was determined as the difference between currents recorded in the presence and absence of extracellular Ca2+. Switches between 2 and 0 mM Ca2+ were made with the rapid solution changer. Peak cell shortening was measured as the difference between peak shortening and a reference point immediately preceding the onset of cell shortening. The amplitudes of Ca2+ transients were measured, after conversion to Ca2+ concentration, as the difference between peak and immediately preceding Ca2+ concentrations.
Statistics. Data are presented as means ± S.E.M. Differences between means were assessed either with a Student's t test, or with a one-way repeated measures analysis of variance. Differences were considered significant for values of p < 0.05. Curve fitting and statistical analyses were performed with Sigmaplot (version 5.0) and SigmaStat (version 2.03) (SPSS Science, Chicago, IL).
Chemicals. Lidocaine, choline chloride, HEPES buffer, EGTA, MgCl2, and analogs of cAMP (Na cAMP, Tris-cAMP, 8-bromo-cAMP, and dibutyryl cAMP) were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). 3-Isobutyl-1-methylxanthine (IBMX) was purchased from Sigma-Aldrich Canada Ltd. Fura-2/AM was purchased from Molecular Probes (Eugene, OR). All other chemicals were purchased from BDH Inc. (Toronto, ON, Canada). A stock solution of Fura-2/AM was prepared by dissolving 50 µg of Fura-2/AM in 20 µl of anhydrous dimethyl sulfoxide (Sigma-Aldrich Canada Ltd.). All other chemicals were dissolved in deionized water.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). In these experiments, test steps were preceded by
10 conditioning pulses from the holding potential of –80 to 0 mV.
Conditioning pulses were followed by 200-ms test steps from a postconditioning
potential of –70 mV to more depolarized membrane potentials.
Figure 1, A and B, shows
representative records of contractions and currents elicited by test steps to
0 and +70 mV in myocytes dialyzed with 50 µM dibutyryl-cAMP. Large
contractions occurred with both test steps, although inward current was large
only with the step to 0 mV. Mean contraction-voltage and I-V relations for
this series of experiments are shown in
Fig. 1, C and D. Although I-V
relations for ICa-L were bell-shaped, contraction-voltage relations
were sigmoidal in the cells dialyzed with dibutyryl-cAMP. Thus, large
contractions were observed with either large or small inward currents when
dibutyryl-cAMP was added to patch pipette solutions.
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The next series of experiments examined contraction-voltage and I-V
relations in myocytes dialyzed with intracellular solution containing the
PDE-sensitive analog Tris-cAMP (Meyer and
Miller, 1974). Representative traces are shown in
Fig. 2, A and B. Currents and
contractions were maximal with a step to 0 mV, whereas both current and
contractions were small with a step to +70 mV.
Figure 2, C and D, shows mean
contraction-voltage and I-V relations for myocytes dialyzed with 50 µM
Tris-cAMP. Contraction-voltage relations were bell-shaped and roughly
proportional to ICa-L with this analog.
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The next series of experiments was designed to determine with the ability of small currents to elicited large contractions was a function of the PDE resistance of the cAMP analog added to the patch pipette solution. To test this, cells were activated with a voltage-clamp protocol consisting of sequential test steps from –65 to –40 mV and then –40 to 0 mV. This protocol was used because the step to –40 mV elicits very little inward current, whereas the step to 0 mV elicits near maximal ICa-L. In addition, to determine whether changes in contraction reflected changes in Ca2+ release, cells were loaded with fura-2 and Ca2+ transients were measured. Figure 3 shows representative recordings of Ca2+ transients and currents in cells dialyzed with either dibutyryl-cAMP or Tris-cAMP. In the presence of PDE-resistant dibutyryl-cAMP, prominent Ca2+ transients were observed with the step to –40 mV, as well as the step to 0 mV (Fig. 3A). In contrast, when myocytes were dialyzed with PDE-sensitive Tris-cAMP, the first activation step did not initiate a transient, although the step to 0 mV initiated a Ca2+ transient and ICa-L (Fig. 3B).
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Figure 4 compares the mean amplitudes of Ca2+ transients and currents recorded in myocytes dialyzed with patch pipettes containing either no cAMP or selected PDE-resistant or PDE-sensitive analogs of cAMP. Figure 4A shows mean Ca2+ transients initiated by the step from –65 to –40 mV. In the absence of cAMP in the patch pipette solution, there was minimal release of Ca2+ with the step to –40 mV. However, in the presence of PDE-resistant 8-bromo or dibutyryl-cAMP, large transients were elicited by this step. In contrast, neither Tris- nor Na cAMP, both of which are PDE-sensitive, supported activation of Ca2+ transients by the step to –40 mV. Interestingly, inward currents elicited by this step were small with all analogs of cAMP tested (Fig. 4B). Figure 4, C and D, shows effects of different cAMP analogs on the magnitudes of Ca2+ transients and currents initiated by a step from –40 to 0 mV, which activates large inward ICa-L. This step initiated Ca2+ transients of similar magnitude regardless of whether cells were dialyzed with cAMP, or whether the cAMP was PDE-sensitive or PDE-resistant. It is possible that the absence of significant effects on transients initiated by steps to 0 mV, even by the PDE-resistant analogs of cAMP, may reflect the order of activation. The large increase in the transient at –40 mV observed with 8-bromo- and dibutyryl-cAMP may have partially depleted the SR and reduced the effect on contractions initiated by the subsequent step to 0 mV. However, the amplitude of ICa-L was increased slightly by all cAMP analogs except Tris-cAMP.
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As shown in Fig. 4, A and B, large Ca2+ transients were elicited by steps to –40 mV, which activated small inward currents only in cells dialyzed with PDE-resistant analogs of cAMP. If PDE resistance accounts for the differences between analogs of cAMP, inhibition of PDE should eliminate this difference. Therefore, we examined the effect of Na cAMP in the presence of IBMX, a nonselective PDE inhibitor. Figure 5, A and B, shows representative recordings of Ca2+ transients and currents activated by steps to –40 and 0 mV in cells dialyzed with Na cAMP. Figure 5A was recorded in the absence of IBMX and shows that a large transient was elicited only by the step to 0 mV. However, when the cell was superfused with 100 µM IBMX (Fig. 5B), a large Ca2+ transient also occurred with the step to –40 mV.
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To determine whether IBMX also would allow endogenous cAMP to activate Ca2+ release at negative potentials we examined the effects of IBMX when cAMP was absent from the pipette solution. Figure 5, C and D, shows recordings of Ca2+ transients and currents from a myocyte dialyzed with intracellular solution with no cAMP added. Figure 5C shows that the step to –40 mV did not elicit a Ca2+ transient; however, the step to 0 mV resulted in a large Ca2+ transient. When the same myocyte was exposed to 100 µM IBMX (Fig. 5D), both steps initiated large Ca2+ transients.
Figure 6 shows mean data for transients and currents with and without IBMX. The mean amplitudes of Ca2+ transients elicited by steps to –40 mV were very small in cells dialyzed either with Na cAMP or with no cAMP in the absence of IBMX (Fig. 6A). However, IBMX caused large increases in the magnitude of Ca2+ transients initiated by steps to –40 mV in cells with Na cAMP or no cAMP. IBMX had no effect on inward current elicited by steps to –40 mV (Fig. 6B), but caused moderate increases in ICa-L activated by steps to 0 mV (Fig. 6D). IBMX had little effect on Ca2+ transients initiated by the step to 0 mV (Fig. 6C). These observations suggest that the absence of Ca2+ transients at negative potentials in cells dialyzed with PDE-sensitive cAMP or no cAMP likely reflects degradation of cAMP by PDE.
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When degradation of cAMP by PDE is minimized, cAMP alters excitation-contraction coupling so that large Ca2+ transients can be elicited with little inward current. It is possible that PDE-resistant analogs of cAMP increase the sensitivity of CICR so that small currents initiate large Ca2+ transients. To determine whether PDE-resistant 8-bromo-cAMP sensitizes CICR, we examined the effects of decreasing the magnitude of ICa-L through partial block with Cd2+. Figure 7 shows the effects of different concentrations of Cd2+ on currents and contractions elicited by sequential test steps to –40 and 0 mV in a cell dialyzed with 8-bromo-cAMP. The voltage protocol, shown at the top of Fig. 7, incorporated a 4-s-long postconditioning potential to allow rapid application of Cd2+ after conditioning pulses but before the test steps. Figure 7A shows that Cd2+ caused a concentration-dependent inhibition of ICa-L elicited by the step to 0 mV. Progressive inhibition of ICa-L was accompanied by a parallel depression of contractions elicited by this step. Thus, CICR clearly was graded by the amplitude of ICa-L in cells dialyzed with PDE-resistant cAMP. However, contractions elicited by the step to –40 mV were not affected by any of the concentrations of Cd2+ tested.
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Figure 7B shows mean
amplitudes of contractions and inward currents plotted as a function of
Cd2+ concentration. Increasing concentrations of Cd2+
caused progressive inhibition of peak ICa-L elicited by steps to 0
mV. The mean amplitude of contractions initiated by the same steps decreased
in parallel with the amplitudes of ICa-L. Thus, small currents only
elicited small contractions. In contrast, the amplitudes of contractions
initiated by the steps to –40 mV were unaffected by Cd2+. It
is unclear whether the small current associated with this step was blocked by
Cd2+, because of the small size of the current and because
Cd2+ blockade is known to be voltage-dependent
(Lansman et al., 1986).
It is possible that the different effects of Cd2+ on the contractions elicited by the steps to –40 and 0 mV might reflect the order of activation. The first step to –40 mV may partially deplete SR stores of Ca2+ and alter the sensitivity of CICR during the subsequent step to 0 mV. We tested this possibility directly by performing similar experiments with a protocol modified to omit the first step to –40 mV as shown at the top of Fig. 8. Figure 8A shows that increasing concentrations of Cd2+ still caused parallel inhibition of ICa-L and contraction with this protocol. Figure 8B presents mean data showing that ICa-L and contraction exhibited essentially identical concentration-response curves to increasing concentrations of Cd2+. These results indicate that PDE-resistant cAMP does not sensitize CICR so that small currents give rise to large contractions or transients at 0 mV.
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Although Cd2+ caused a graded block of CICR at 0 mV, large
contractions persisted at –40 mV in the presence of cAMP. The gain of
CICR has been reported to increase at negative membrane potentials
(Lopez-Lopez et al., 1995;
Santana et al., 1996;
Adachi-Akahane et al., 1999).
Thus, it is possible that PDE-resistant cAMP sensitizes CICR at negative
potentials where gain is already high. Beuckelmann and Wier
(1988) reported that
repolarization to negative potentials from positive potentials induces
contractions attributable to CICR. The magnitude of ICa-L elicited
by repolarization can be graded by the degree of inactivation caused by the
initial depolarization. We used this approach to examine the relationship
between amplitude of ICa-L and contraction at –40 mV in cells
dialyzed with PDE-resistant cAMP. Figure
9 shows the voltage-clamp protocol plus representative recordings
of currents and contractions. Cells were depolarized from –65 mV to a
potential between 0 and +100 mV, followed by repolarization to –40 mV.
The amplitudes of contractions initiated by repolarization varied with the
membrane potential during the preceding depolarization. Contractions were
large when the preceding depolarization was to +100 mV, but decreased to
negligible levels as depolarization approached 0 mV. Inward currents elicited
by repolarization were obscured by the capacitive transient caused by this
voltage step. To measure inward Ca2+ currents initiated by
repolarization, the same protocols were repeated in extracellular solution
containing 0 mM Ca2+. Recordings in 0 mM Ca2+ were
subtracted from recordings made in 2 mM Ca2+ to provide the
recordings of ICa-L shown in
Fig. 10A. The amplitude of
peak inward Ca2+ current was maximal when the preceding
depolarization was to +100 mV and decreased to negligible levels as
depolarization approached 0 mV. Figure
10B shows records of contraction accompanying these inward
currents. Figure 10C shows a
plot of contraction amplitude as a function of the magnitude of inward current
for the traces shown in Fig. 10, A and
B. Contraction was directly proportional to the amplitude of
ICa-L.
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Figure 11, A and B, shows mean amplitudes of contractions and currents as functions of prepulse voltage, for five replicates of the experiment illustrated in Fig. 10. Both contraction and current increased with the voltage of the prepulse. Figure 12A shows a plot of mean amplitudes of contraction as a function of mean peak inward ICa-L from the mean data shown in Fig. 11. The mean data were well fitted by a regression line (r2 = 0.99). Figure 12A also shows 95% confidence limits for this regression. These data clearly show that contractions remained proportional to ICa-L at –40 mV in the presence of PDE-resistant 8-bromo-cAMP. Thus, in protocols designed to examine CICR directly small currents elicited only small contractions, even at negative potentials where the gain of CICR is high.
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Figure 12B shows a similar plot for mean amplitudes of contraction and peak inward ICa-L for experiments in which the magnitude of ICa-L was graded by increasing concentrations of Cd2+ (Fig. 7). Figure 12B shows that contraction also was directly proportional to peak ICa-L in protocols in which ICa-L was elicited by depolarization rather than repolarization. Figure 12, A and B, also shows data for mean contractions and currents elicited by depolarizing steps from –65 to –40 mV in the same experiments. These steps elicited large contractions with little inward current. These data clearly do not fall within the confidence limits for the relationships between contraction and current for conventional CICR in Fig. 12, A and B. Our observations demonstrate that PDE-resistant cAMP does not sensitize conventional CICR so that small currents trigger large contractions.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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In the present study, all experiments were conducted with 0 mM
Na+ in the patch pipette solution to inhibit influx of
Ca2+ by way of Na+-Ca2+ exchange. Under these
conditions, the occurrence of bell-shaped and sigmoidal contraction-voltage
relations depended on whether myocytes were dialyzed with PDE-sensitive or
PDE-resistant analogs of cAMP. This difference may explain observations made
in previous studies. We and others
(Ferrier et al., 1998;
Mackiewicz et al., 2000) have
reported that intracellular dialysis with 8-bromo-cAMP results in sigmoidal
contraction-voltage relations. The present study showed the same result with
another PDE-resistant analog of cAMP, dibutyryl-cAMP. On the other hand,
Piacentino et al. (2000)
reported bell-shaped contraction-voltage relations when cells were dialyzed
with Tris-cAMP. The present study agrees with this finding and shows that a
bell-shaped contraction-voltage relationship would be expected in cells
dialyzed with Tris-cAMP, which is readily broken down by PDE. Thus, the
occurrence of sigmoidal or bell-shaped contraction-voltage relations likely
reflects the use of PDE-resistant or PDE-sensitive cAMP analogs,
respectively.
To investigate whether PDE-resistant cAMP modified CICR so that small
ICa-L currents elicited large responses, we used two protocols that
clearly activated CICR. First, CICR responses initiated by voltage-clamp steps
from –40 to 0 mV were examined. The magnitude of ICa-L
initiated by this step was graded by different concentrations of
Cd2+. With this approach, we found that contractions were smoothly
graded by the amplitude of ICa-L, and small ICa-L
currents initiated only very small contractions. Large contractions initiated
by conventional CICR occurred only in response to large currents. These
experiments suggest that sensitization of CICR by cAMP cannot explain the
large contractions accompanied by small inward currents at more negative
potentials (e.g., –40 mV). However, the gain of CICR is greater at
negative potentials than at more depolarized potentials
(Lopez-Lopez et al., 1995;
Santana et al., 1996;
Adachi-Akahane et al., 1999).
Thus, it is possible that sensitization of CICR would only be observed at more
negative potentials. To test this possibility, ICa-L was initiated
by repolarization to –40 mV
(Barcenas-Ruiz and Wier, 1987).
The repolarizing step was made from different positive potentials to vary the
magnitude of ICa-L through partial inactivation. These experiments
demonstrated that, even at –40 mV, CICR remained graded by magnitude of
current so that small ICa-L currents elicited only very small
contractions. Thus, dialysis of cells with PDE-resistant cAMP did not alter
CICR so that small ICa-L currents would initiate large contractions
at either 0 or –40 mV.
The mechanism by which large contractions and Ca2+ transients
can be initiated with little current in cells dialyzed with PDE-resistant cAMP
is not clear. High-gain CICR has been suggested as one possibility
(Hussain and Orchard, 1997;
Wier and Balke, 1999;
Piacentino et al., 2000).
However, our experiments show that CICR coupled to whole cell ICa-L
cannot account for this high-gain mechanism. We have observed large responses
accompanied by little inward current when activation steps are made from
potentials more negative than –40 mV
(Ferrier et al., 1998;
Howlett et al., 1998;
Ferrier and Howlett, 2001).
Thus, one might propose that high-gain CICR is coupled to T-type
Ca2+ current, which activates at negative membrane potentials
(McDonald et al., 1994).
However, T-current has been reported to be a weak trigger for CICR
(Sipido et al., 1998), and
most studies suggest that T-current is insensitive to elevation of cAMP
(McDonald et al., 1994). This
does not preclude the possibility that the efficacy of T-current to increase
SR Ca2+ release might be increased by phosphorylation of ryanodine
receptors, because protein kinase A-dependent phosphorylation has been shown
to increase the ligand sensitivity of ryanodine receptors
(Uehara et al., 2002). Another
possibility is that high-gain CICR is coupled to Ca2+ entry through
sodium channels (slip-mode conductance)
(Santana et al., 1998).
However, contractions initiated at negative potentials persist in the presence
of 50 µM tetrodotoxin (Ferrier et al.,
2000), which strongly inhibits Ca2+ influx via this
means (Santana et al., 1998).
Alternatively, after phosphorylation high gain CICR might be coupled to a
subpopulation of L-type Ca2+ channels. This subpopulation of
channels must have distinct properties, because activation must occur at very
negative membrane potentials and little inward current is observed. However, a
mechanism coupled to L-type Ca2+ current should be activated by
current initiated either by depolarization or by repolarization
(Beuckelmann and Wier, 1988),
which was not the case. Therefore, if this mechanism is coupled to a
subpopulation of L-type Ca2+ channels, these channels must exhibit
rapid voltage-inactivation with strong depolarizations, to account for the
observation that this mechanism is not activated upon repolarization.
Alternatively, because this mechanism is only activated on depolarization and
not repolarization, one cannot exclude the possibility of a mechanism that
operates independently of Ca2+ current on the basis of the data
presented here (Beuckelmann and Wier,
1988). Additional investigations will be needed to explore these
possibilities.
Our results demonstrate that dialysis of myocytes with PDE-resistant cAMP
changes the configuration of the contraction-voltage relationship. A change in
the configuration of the contraction-voltage relation could alter the efficacy
of the action potential as a trigger for Ca2+ release. The action
potential of mammalian ventricular cells typically reaches approximately +40
mV during the upstroke. This voltage is well beyond the peak of the I-V
relationship for ICa-L. Thus, Ca2+ release and
contraction would not be maximal if controlled by the bell-shaped curve
observed without cAMP dialysis or PDE inhibitors. However, in the presence of
PDE-resistant cAMP or PDE inhibition, a maximal contraction would be generated
at these positive potentials. Thus, the change in the configuration of the
contraction-voltage relation may contribute substantially to the positive
inotropic effects of agents that increase cAMP, such as 1 agonists.
cAMP only affected the configuration of contraction-voltage relations if
the analog was PDE-resistant or if PDE was inhibited. Our results further show
that even in the absence of exogenous cAMP, inhibition of PDE by IBMX can
modulate Ca2+ release. This observation suggests that basal cAMP
synthesis is active even in the absence of 1 agonists. However, much of
this cAMP must be hydrolyzed by PDE because SR Ca2+ release is only
stimulated if cAMP degradation is inhibited. Interestingly, in a previous
study we found that amrinone, a PDE III inhibitor, increased contraction with
little effect on ICa-L (Xiong
et al., 2001). Collectively, these observations suggest that PDE
may serve an important regulatory role in cardiac excitation-contraction
coupling.
PDE-resistant cAMP and IBMX caused only modest increases in
ICa-L and little, if any, increase in Ca2+ transients
initiated by steps to 0 mV. This further indicates that cAMP does not
sensitize CICR. A similar result has been observed with the L-channel agonist
Bay K 8644. Bay K 8644 increases ICa-L but not contraction
(McCall and Bers, 1996;
Adachi-Akahane et al., 1999).
It is believed that this occurs because Bay K 8644 increases ICa-L
by increasing the duration of single channel openings, not the number of
channels which open (Bers,
2001). Because a single channel opening may initiate release
(Lopez-Lopez et al., 1995;
Santana et al., 1996;
Wang et al., 2001), increasing
the duration of openings would increase current but would not activate any
further release. Interestingly, 8-bromo cAMP or stimulation of
-adrenergic receptors both cause a similar prolongation of single
channel open times (Yue et al.,
1990). This may explain why CICR transients were not increased in
the present study despite a modest increase in peak inward
ICa-L.
In summary, the present study demonstrates that intracellular dialysis with cAMP can result in large contractions and Ca2+ transients occurring with little inward current. However, this only occurs if degradation of cAMP is restricted by use of a PDE-resistant analog or by treating the cells with a PDE inhibitor. Nonetheless, CICR remains smoothly graded by the amplitude of ICa-L, with small currents eliciting only small responses. Our results also indicate that PDE may serve an important regulatory role in excitation-contraction coupling by modulating levels of cAMP. Changes in cAMP levels may alter contraction by changing the relationship between Ca release and membrane potential.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: ICa-L, L-type Ca2+ current; SR, sarcoplasmic reticulum; CICR, Ca2+-induced Ca2+ release; I-V, current-voltage; PDE, phosphodiesterase; IBMX, 3-isobutyl-1-methylxanthine; Bay K 8644, S(–)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid methyl ester.
Address correspondence to: Dr. Susan E. Howlett, Department of Pharmacology, Sir Charles Tupper Medical Bldg., Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7. E-mail: susan.howlett{at}dal.ca
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