© 1995 American Heart Association, Inc.
Articles |
cGMP-Dependent Protein Kinase Regulation of the L-Type Ca2+ Current in Rat Ventricular Myocytes
From the Department of Molecular and Cellular Physiology, University of Cincinnati (Ohio).
Correspondence to Dr Nicholas Sperelakis, Department of Molecular and Cellular Physiology, University of Cincinnati, PO Box 670576, Cincinnati, OH 45267-0576.
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Abstract |
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Abstract Regulation of L-type Ca2+ channel current [ICa(L)] by cGMP-dependent protein kinase (PK-G) was investigated in ventricular myocytes from 2- to 21-day-old rats using whole-cell voltage clamp with internal perfusion. ICa(L) was elicited by a depolarizing pulse to +10 mV from a holding potential of -40 mV. Stimulated ICa(L) (by 2 µmol/L isoproterenol) was inhibited to the basal level by internal perfusion with 50 nmol/L PK-G (activated by 8Br-cGMP, 0.1 µmol/L). When ICa(L) was enhanced by Bay K 8644 (1 µmol/L), the enhanced basal ICa(L) was also reduced by PK-G. Basal ICa(L) (nonstimulated through the cAMP/cAMP-dependent protein kinase [PK-A] pathway) was also inhibited to various degrees (large, medium, or small) by internal application of PK-G (25 nmol/L). The average inhibition was 42.1% (n=36), and there were no differences in the inhibition during development. The inhibition by PK-G was blocked by the PK-G substrate peptide (cG-PKI, 300 µmol/L) and by heat inactivation of the PK-G. Relatively specific PK-G inhibitors (eg, cG-PKI and H-8) sometimes reversed the inhibition (5 of 25 cells), whereas isoproterenol stimulated ICa(L) (7 of 8 cells). When a holding potential of -80 mV was used, the inhibition produced by PK-G was much less. The inhibitory effects of PK-G were not mediated by activating phosphodiesterase or protein phosphatase but most likely by a direct phosphorylation of the Ca2+ channel or associated regulatory protein. The inhibitory effect of PK-G may be explained by a balance between activities of PK-A and PK-G in regulating the slow Ca2+ channels at two separate sites.
Key Words: Ca2+ slow channels • whole-cell voltage clamp • patch clamp • cGMP-dependent protein kinase • internal perfusion technique
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Introduction |
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The voltage- and time-dependent slow (L-type) Ca2+ channels in the cell membrane are the major pathway by which Ca2+ ions enter the cell during excitation for initiation and regulation of the force of contraction of the muscle. The channel can be controlled by extrinsic factors (such as autonomic nerve stimulation or circulating hormones) and by intrinsic factors (such as cellular pH or ATP level). One major mechanism for regulation of the L-type Ca2+ channels is elevation of cAMP level and stimulation of PK-A phosphorylation of the channel or a closely associated regulatory protein. Such phosphorylation results in an increase in the open probability of the channel1 2 and in the enhancement of peak amplitude of ICa(L).3
It has been proposed that cGMP plays a role antagonistic to
that of cAMP.4 The antagonism produced by cGMP/PK-G was
reported to involve the direct phosphorylation of the
Ca2+ channel or a closely associated regulatory protein by
PK-G in guinea pig and rat ventricular
cells.5 6 7 8 9 It was reported that a single protein of 
47
kD is specifically phosphorylated by PK-G (in the
presence of 10-5 mol/L cGMP) in guinea pig sarcolemmal
preparations,10 and this protein may be a possible
mediator involved in regulation of Ca2+ channels of the
heart by the cGMP/PK-G pathway. cGMP was also reported to increase cAMP
degradation by a cGMP-stimulated PDE (PDE-II) in frog
ventricular myocytes.11 12 It has been
reported that under some conditions, cGMP may actually further
stimulate the prestimulated ICa(L) rather than inhibit it,
presumably by preventing cAMP degradation by cGMP inhibition of a
phosphodiesterase (PDE-III).13
The effect of cGMP on basal ICa(L) (not stimulated through the cAMP/PK-A pathway) is still controversial. Although some investigators reported that there were no effects of cGMP on basal ICa(L),9 10 11 12 13 we have evidence that cGMP does have an inhibitory effect on basal ICa(L). Intracellular application of cGMP (by pressure injection,5 by the liposome method,14 or by using the membrane-permeable derivative 8Br-cGMP)6 8 inhibited the Ca2+-dependent slow action potentials in guinea pig papillary muscles and chick embryonic heart cells. We also reported that 8Br-cGMP inhibited basal ICa(L) in chick embryonic heart cells by using whole-cell voltage-clamp8 and cell-attached patch-clamp15 techniques. Recently, we found that in embryonic heart cells, intracellular application of PK-G via the patch pipette inhibited the basal and the prestimulated ICa(L)s.16
In the present study, whole-cell voltage clamp (with internal perfusion technique) was performed on young rat ventricular myocytes to examine the effect of PK-G on ICa(L). We found that PK-G inhibited both the basal ICa(L) as well as the prestimulated ICa(L).
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Materials and Methods |
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Cell Preparation
Freshly isolated single cells were prepared from ventricles of young rats (Sprague-Dawley) as previously described.17 The rats were divided into four age groups: 3-day (2- to 4-day-old), 7-day (6- to 8-day-old), 11-day (10- to 13-day-old), and 20-day (19- to 21-day-old) rats. The rats were anesthetized by exposing them to an atmosphere of pure CO2 for a few minutes and then decapitated. The hearts were dissected and rinsed in oxygenated normal Tyrode's solution and then immersed in nominally Ca2+-free Tyrode's solution.17 The ventricles were dissected from the atria after the spontaneous contractions had ceased (
15 minutes), and
small pieces of ventricular tissue were enzymatically
digested for 50 minutes (at 37°C) in the Ca2+-free
Tyrode's solution containing 1 mg/mL collagenase (Yakult).
After incubation, the tissues were rinsed at least three times in
modified KB solution17 at room temperature, and the cells
were mechanically dispersed with a wide-bore pipette. The cell
suspension was stored at 4°C in the modified KB solution. Cells were
used between 2 and 6 hours after isolation.
Solutions and Drugs
The Na+-free and K+-free external
solution contained (mmol/L)
tetraethylammonium chloride 150,
CaCl2 1.8, MgCl2 0.5, 4-AP 3, HEPES 5, and
glucose 5.5 (pH 7.4 using HCl), so as to isolate the Ca2+
current from the Na+ and K+ currents. The
internal (pipette) solution contained (mmol/L) CsOH 110, CsCl 20,
L-glutamic acid 90, MgCl2 3,
ATP-Na2 5, phosphocreatine disodium salt 5, EGTA 10, and
HEPES 5 (pH 7.2 with CsOH).
ISO, H-8, and Rp-8-pCPT-cGMPS were dissolved in distilled water to provide a stock solution (1 mmol/L, 5 mmol/L, and 5 mmol/L, respectively). Bay K 8644 and MC were dissolved in ethanol to provide 10 mmol/L and 1 mmol/L stock solutions, respectively. IBMX was dissolved in DMSO to provide a 100 mmol/L stock solution. ISO, H-8, Rp-8-pCPT-cGMPS, Bay K 8644, and IBMX were diluted to appropriate concentrations and applied extracellularly. It was confirmed that the solvents used, ethanol (up to 0.05%) and DMSO (up to 0.1%), did not affect ICa(L). PK-G, cG-PKI, and MC were directly dissolved in the internal solution for intracellular application at a final concentration of 25 or 50 nmol/L for PK-G, 300 µmol/L for cG-PKI, and 0.5 µmol/L for MC. The PK-G solution was always accompanied by a potent and unhydrolyzable PK-G activator, 8Br-cGMP (0.1 µmol/L). PK-G and cG-PKI were stored at 4°C and used within 3 weeks. The sources of these drugs were as follows: ISO, H-8, 8Br-cGMP, IBMX, and MC from Sigma Chemical Co, PK-G and cG-PKI from Promega, Rp-8-pCPT-cGMPS from BioLog Life Science Institute, and Bay K 8644 from Dr Alexander Scriabine (Miles Laboratory).
Whole-Cell Voltage-Clamp Recording
Whole-cell voltage-clamp recordings, with the
perfusion pipette technique, were made by using an EPC-7
patch-clamp amplifier (List-Electronic). The patch pipettes had
resistances of 1.5 to 3 M
(when filled with the internal solution).
Series resistance was partly compensated electrically (10% to 30%).
The cells were placed into a perfusion chamber (which contained the
external solution) located on an inverted microscope (Nikon) and
allowed to settle for at least 5 minutes. Then they were constantly
perfused with the external solution at 3 mL/min at room temperature
(22°C to 25°C). All solutions containing drugs or proteins were
applied extracellularly and/or intracellularly after the
ICa(L) had stabilized (usually 3 minutes after breaking
into the cell and obtaining the whole-cell configuration). The
liquid junction potential between the internal solution and the
external solution was -2.0±0.2 mV (mean±SEM, n=7). This value was so
small and negligible that potentials given in the data were not
corrected for the junction potential.
The ICa(L) currents were recorded at 0.1 Hz from an HP of -40 mV (pulses of 300-millisecond duration), unless otherwise mentioned, to exclude any ICa(T) or the possibility of Ca2+ traversing the fast Na+ channel. The currents were abolished completely by 2 mmol/L Co2+ or 0.5 mmol/L Cd2+, consistent with the current being carried through a Ca2+ channel. To see the effect of the PK-G, peak ICa(L) was generated with pulses to +10 mV. Current-voltage curves were obtained by applying voltage steps (300 milliseconds in duration) in 10-mV increments (-30 to +70 mV) from an HP of -40 mV. The inactivation curve was determined by using a standard double-pulse protocol: 3000-millisecond conditioning pulses of various amplitudes (from an HP of -40 mV) were followed by a test pulse to 10 mV (300 milliseconds in duration). A 5-millisecond gap was set between the conditioning pulse and test pulse (to -40 mV) to allow for resetting of the activation gate.
Leak and capacitance currents were subtracted by using currents elicited by small hyperpolarizing pulses (P/5 protocol), unless otherwise mentioned. Current signals were filtered with a cutoff frequency of 1 kHz (eight-pole Bessel) and sampled at 3 kHz. Current and voltage signals were stored on an IBM-AT–compatible personal computer; the PCLAMP program (Axon Instruments) was used for further analysis. Current density was calculated from the measured membrane capacitance. Membrane capacitance was determined by applying ramp voltage pulses (-0.5 V/s) from an HP of 0 mV; no ionic currents were activated during the voltage pulses.
Internal Perfusion Technique
PK-G and/or cG-PKI was applied intracellularly via the pipette
by using a modified internal perfusion technique devised by Kameyama et
al.3 A thin quartz tubing (Adams & List Associates) was
connected with a long (80 cm) thin (internal diameter, 0.28 mm)
polyethylene tubing (Clay Adams) and inserted into the glass pipette so
that the tip of the quartz tubing was close to the pipette tip. A
change of solution in the pipette (perfusion rate, 4 to 6 µL/min) was
carried out by applying a negative pressure to the back end of the
glass pipette. The perfusion quartz tubing was first filled with the
control pipette solution to 10 cm from the tip to prevent premature
diffusion into the pipette tip and the cell interior.
A tiny bubble (1 to 1.5 mm) was inserted into the thin tubing to
separate the control internal solution and the subsequent experimental
solution (containing the test substance). When two agents were to be
internally perfused sequentially, another bubble was added for
separation. The bubbles were useful to identify the change of solution,
to control speed of perfusion by optically watching their movement, and
to reduce the electrical noise pickup (60 Hz). In control experiments,
the tiny bubble did not alter the peak amplitude of ICa(L)
that was recorded when it reached the end of the quartz tubing
(n=8, data not shown). Before perfusion was started, the polyethylene
tubing was completely clamped in the middle by a string attached to a
manipulator. The efficacy of the internal perfusion technique was
checked by observing an increase in ICa(L) with cAMP (100
or 500 µmol/L) added to the pipette as a stimulatory agent (n=4, data
not shown).
Data Analysis
Current amplitudes were measured as the peak inward current.
Experiments were discarded if the analyzed time course clearly
showed substantial rundown during the control period before application
of PK-G.
The dose-response curve was fitted by the Hill equation:
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The inactivation curves were fitted using the conventional Boltzmann
equation:
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All data are presented as mean±SEM with the number of cells in parentheses. Statistical evaluation was made by the paired t test for PK-G effect and by ANOVA for comparing more than three groups. A value of P<.05 was considered to be statistically significant.
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Results |
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PK-G Inhibition of Prestimulated ICa(L)
ICa(L) of rat ventricular myocytes was prestimulated by 2 µmol/L ISO, and the effect of PK-G (50 nmol/L) was examined. The concentration of 2 µmol/L was reported to exert almost maximum stimulatory effect in newborn and adult rabbit and rat ventricular cells.18 19 We first checked the stimulatory effect of ISO (2 µmol/L) on ICa(L). The average stimulation by ISO was 83.4±14.9% for myocytes from the 3-day (n=4), 110±25.5% for 11-day (n=4), and 112±17.5% for 20-day (n=8) groups. ISO could maintain a sustained stimulatory effect for up to 15 to 20 minutes (n=3; Fig 1A
, inset). After
ICa(L) stimulation reached maximum, PK-G (50 nmol/L) was
perfused intracellularly via the perfusion patch pipette. A low
concentration of 8Br-cGMP (0.1 µmol/L) always accompanied the
PK-G in order to activate its enzymatic activity (since there
is no catalytic subunit). Fig 1A shows a typical experiment. As can be
seen, prestimulated ICa(L) was inhibited to about the basal
level by PK-G application within 9 minutes after the start of
perfusion. Similar results were obtained in a total of six cells in
variable stages of development (n=2 for 3-day, n=1 for 11-day, and
n=3 for 20-day groups). The steady state level of inhibition, which was
almost the same as or lower than the control level (-9.2±13% from
the control level, n=6), was reached within 4 to 10 minutes after PK-G
application. After PK-G perfusion was stopped, no recovery was evident
within 5 minutes (n=4, data not shown).
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A second method of stimulating ICa(L), which does
not involve the cAMP/PK-A pathway, was also used. ICa(L)
was stimulated by the Ca2+ channel agonist Bay K 8644 (1
µmol/L). A representative experiment is shown in Fig 1B
. PK-G (50 nmol/L) reversed the Bay K 8644 stimulation of
ICa(L) back to the control level within 3 minutes after
PK-G application. Similar experiments were obtained in a total of three
cells. The decrease in ICa(L) reached a steady state level,
which was lower (-28.2±15.2%, n=3) than the control level, within 3
to 5 minutes after PK-G application. One control experiment showed that
the enhancement produced by Bay K 8644 was maintained for 5 minutes
(Fig 1B, inset). Therefore, the inhibitory effect produced
by PK-G on ICa(L) was not caused by desensitization to Bay
K 8644. Since Bay K 8644 has been reported not to increase cAMP
levels20 or to activate endogenous
protein kinase activity in the heart,21 the
inhibitory effect of PK-G on Bay K 8644–enhanced
ICa(L) may reflect the endogenous activity of
PK-A.
PK-G Inhibition of Basal ICa(L)
Similar experiments were performed on the basal (not stimulated
through the cAMP/PK-A pathway) ICa(L). Since a low dose
(0.1 µmol/L) of the potent and unhydrolyzable PK-G
activator, namely 8Br-cGMP, was present to
activate the enzymatic activity, we first tested its effect.
8Br-cGMP itself had only a slight inhibitory effect on
basal ICa(L) (average inhibition, 10.5±1.9%; n=8; Fig 2). However, when 25 nmol/L PK-G was perfused together
with the 8Br-cGMP, ICa(L) was markedly inhibited: by
47.2±8.6% from the control level (n=5, Fig 2A). As expected, higher
doses of 8Br-cGMP (1 or 10 µmol/L) caused substantial inhibition
of the basal ICa(L) (Fig 2B). The steady state level of
inhibition was reached within 6 minutes after PK-G application. As can
be seen in Fig 2A (inset), after the application of PK-G, a small
outward current sometimes appeared. The calculated values of
IC50, the Hill coefficient, and percent inhibition
(maximum) were 0.29 µmol/L, 1.32, and 52.5%, respectively (Fig 2B).
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The leak-subtracted current-voltage curves for basal
ICa(L) before and after application of 25 nmol/L PK-G
(n=11) are summarized in Fig 3A. PK-G produced a
decrease in ICa(L) at all test potentials without changing
the apparent threshold potential (-30 mV) and potential for peak
current (between +10 and +20 mV). The inhibitory effect was
statistically significant at potentials of +10 mV and higher. At a test
potential of +10 mV, PK-G inhibited the basal ICa(L) by
46.6% (from -8.35±1.94 to -4.46±0.70 pA/pF, n=11). The reversal
potential was shifted to the left by 12 mV after PK-G application.
Typical current traces of ICa(L) before and after
application of PK-G (25 nmol/L) are illustrated in Fig 3B. As shown,
PK-G decreased basal ICa(L) at each test potential. At test
potentials of +50 and +60 mV, a small outward current was produced. The
outward currents were always seen after PK-G application, especially at
test potentials from +50 mV and higher. Preliminary experiments suggest
that this outward current is Cs+-insensitive,
4-AP–insensitive, and
tetraethylammonium-insensitive
K+ current, which is activated by PK-G, because
application of a Cl- channel blocker (SITS, 3 mmol/L;
Sigma) or change to low [Cl]o (15 mmol/L) failed to
diminish the outward current (data not shown). The outward current
persisted even when ICa(L) was blocked by 0.5 mmol/L
Cd2+, thus indicating that the outward current was
not dependent on Ca2+ influx. To prevent any change in the
half-cell potential in the low [Cl]o experiment, an
agar bridge was connected so that the Ag/AgCl electrode was immersed in
a constant Cl- concentration.
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) and after application
of PK-G (
). Currents are normalized as pA/pF. Data points are
shown as mean±SEM. As can be seen, the threshold voltage was 
P<.01. B, Typical current traces for
one cell at different test potentials (+20 to +60 mV) for the control
condition (top traces) and after application of PK-G (bottom traces).
PK-G reduced the basal ICa(L) at all test potentials. At
test potentials of +50 mV and +60 mV, small outward currents
occurred.
Steady state inactivation curves were plotted as current densities (Fig 4A) and as normalized currents (to the maximum
ICa(L) obtained at a conditioning pulse of -80 mV) (Fig 4B). PK-G (25 nmol/L) significantly decreased the current density
between -40 and -20 mV (Fig 4A), and the inhibition was not
significant at -80 and -60 mV (P<.07). The normalized
curves were fitted by the Boltzmann equation (Fig 4B). Calculated
values of the half-inactivation potential and slope factor for the
control curve were -22 and 5.2 mV, respectively, and those for PK-G
were -28 and 6.5 mV, respectively. There was a slight (6-mV) shift in
the hyperpolarizing direction after PK-G application (Fig 4B). The
relative values at a conditioning pulse of -40 mV were 0.95±0.01 for
the control condition and 0.84±0.05 after PK-G application (n=6). This
slight shift in steady state inactivation cannot account for the
overall inhibitory effect.
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Some experiments were performed to examine the reversibility of the PK-G inhibition of basal ICa(L). Once the steady state level of inhibition was attained, PK-G (25 nmol/L) perfusion was stopped by clamping the perfusion tubing (n=12). In most experiments (10 of 12), the inhibitory effect of PK-G was long lasting, and ICa(L) remained inhibited for at least 5 minutes after PK-G perfusion was stopped; however, in two experiments, there was a partial (27% and 33%) recovery of ICa(L) (data not shown).
Mechanism of Inhibition by PK-G on Basal
ICa(L)
To check whether the effect of PK-G on basal ICa(L)
was due to its enzymatic activity, a stock solution containing only
PK-G was incubated in a hot water bath (92°C) for 30 minutes, and
this heated PK-G was used for the internal solution (25 nmol/L plus 0.1
µmol/L 8Br-cGMP). As can be seen in Fig 5A, the
heat-inactivated PK-G had only a slight
inhibitory effect (9.0±2.2%, n=4), which is almost equal
to the effect of the low dose of 8Br-cGMP alone (Fig 2). However,
subsequent addition of intact PK-G produced a much greater inhibition
(35.3±4.9% of control, n=4) of the basal ICa(L) within 5
minutes. Similar results were produced in two other experiments.
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To test whether the inhibitory effect of PK-G was due to
its enzymatic activity, the effects of three different PK-G
inhibitors were examined. Fig 5B shows a
representative experiment in which
simultaneous addition of the PK-G inhibitor,
cG-PKI (300 µmol/L), prevented the effect of 25 nmol/L PK-G. As can
be seen, cG-PKI prevented reduction of ICa(L) by PK-G, and
once this inhibitory peptide was removed (its perfusion
stopped), the effect of PK-G to inhibit ICa(L) was
gradually exerted over the next 3 minutes. Similar results were
obtained in a total of three experiments, and they confirmed that the
inhibitory effect of PK-G was prevented by cG-PKI. These
results suggest that the downregulation of ICa(L) by PK-G
is due to its enzymatic activity.
The mechanism of inhibitory effect of PK-G may be explained by the following three possibilities: (1) direct phosphorylation of the channel or an associated protein, (2) activation of a PDE, or (3) activation of a PPase.
Some experiments were conducted to rule out the second possibility by
using a nonspecific PDE inhibitor, IBMX. Akita et
al18 reported that in rabbit cardiomyocytes at
least 100 µmol/L (adult) and 300 µmol/L (newborn) of IBMX were
required to exert maximum stimulatory effect on basal
ICa(L). Therefore, these two doses of IBMX were tested on
rat heart cells. External application of 100 µmol/L IBMX only
slightly increased the basal ICa(L) (12.0±1.2%, n=4), and
internal application of PK-G (25 nmol/L) markedly inhibited
ICa(L) (Fig 6A). Similar results were
obtained in three cells. In two of the three experiments, there was a
slight recovery after stopping PK-G perfusion (Fig 6A). The higher dose
of IBMX (300 µmol/L) had almost no effect on basal ICa(L)
(-1.6±6.7%, n=7), but internal perfusion of PK-G (25 nmol/L)
inhibited ICa(L) (n=2, Fig 6B). In both experiments,
ICa(L) slightly decreased after stopping the perfusion. As
a control, the ability of IBMX to inhibit PDE was checked by using ISO.
In the presence of IBMX (300 µmol/L), washout of ISO (2 µmol/L) did
not reverse the stimulation within 20 minutes (n=3) (Fig 6B, left
inset), whereas it rapidly reversed in the absence of IBMX (Fig 6B,
right inset). These results indicate that the action of PK-G was not
mediated through PDE activation.
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A nonselective PPase (1 and 2A) inhibitor, MC (0.5
µmol/L) added in the pipette, was used to exclude the third
possibility. MC (0.5 µmol/L) was reported to inhibit PPases in frog
atrial cells.22 After the whole-cell configuration was
formed, ICa(L) increased, reaching a steady level by 4 to 7
minutes (n=7, data not shown). As can be seen in Fig 7,
PK-G (25 nmol/L) was still capable of inhibiting ICa(L) in
the presence of MC (n=5 cells). As a control, the ability of MC to
inhibit PPases was checked by using ISO. In the presence of MC (0.5
µmol/L), washout of ISO (2 µmol/L) did not reverse the stimulation,
and the effect persisted for almost 20 minutes (n=2; Fig 7, inset).
These results indicate that the action of PK-G was not mediated through
PPase activation.
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Variable Inhibitory Effect of PK-G on Basal
ICa(L)
The average basal inhibition of ICa(L) by PK-G was
42.1±2.9% (n=36). However, PK-G (25 nmol/L) produced variable
degrees of inhibition from cell to cell: large (54.5%, Fig 8A), moderate (34.3%, Fig 8B), or small (13.7%, Fig 8C). The time required to reach a stable-state inhibition was 3 to
6 minutes after the start of PK-G application.
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To test whether the variable response to PK-G may be due to
possible developmental changes in the intrinsic PK-A and PK-G
activities, the inhibition of basal ICa(L) by PK-G (25
nmol/L) was compared for rats in the four neonatal stages of
development: 3-day, 7-day, 11-day, and 20-day groups. As shown in Fig 9, there were no statistically significant differences
among the four age groups with respect to the average
inhibitory action of PK-G: 44.0±5.7% (n=7), 42.8±7.5%
(n=7), 39.7±4.2% (n=15), and 44.7±8.7% (n=7), respectively. The
variable degrees of inhibition from cell to cell were also seen in
all four groups (Fig 9).
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Reversibility of the Inhibited ICa(L) by ISO and
PK-G Inhibitors
As already described, simply stopping perfusion of PK-G was
insufficient to reverse the downregulated ICa(L).
Therefore, we checked the reversibility by using ISO and some PK-G
inhibitors. After the PK-G (25 µmol/L) inhibition reached
a steady state level, ISO (2 µmol/L) was applied from outside (in the
11-day group). The average inhibition of basal ICa(L) was
40.1±7.0% (n=7). As shown in Fig 10, ISO could
stimulate the inhibited ICa(L) in seven of the eight cells
tested. These results suggest that upregulation through the PK-A
pathway can still occur in the presence of PK-G inhibition.
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Experiments were also performed in which the effects of PK-G were
attempted to be reversed by application of PK-G inhibitors.
After PK-G (25 nmol/L) reduced ICa(L) to a steady level,
the PK-G inhibitors, cG-PKI (300 µmol/L), H-8 (1-5
µmol/L), and Rp-8-pCPT-cGMPS (2-10 µmol/L), were tested for
recovery of ICa(L). H-8 and Rp-8-pCPT-cGMPS were applied
externally because they are membrane permeable; cG-PKI was perfused
internally. These respective concentrations were used because their
reported Ki values (for respective values of
PK-A versus PK-G) were 480 and 120 µmol/L for cG-PKI,23
1.2 and 0.48 µmol/L for H-8,24 and 8.3 and 0.5 µmol/L
for Rp-8-pCPT-cGMPS (BioLog Co, personal communication, unpublished
data, 1994). In most experiments (20 of 25), cG-PKI, H-8, and/or
Rp-8-pCPT-cGMPS could not reverse the PK-G–inhibited
ICa(L) (Fig 11A and 11B), but sometimes (5
of 25 experiments) they produced some degree of reversibility. As shown
in Fig 11C, one experiment exhibited a full recovery by H-8.
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Voltage Dependence of the PK-G Inhibition of Basal
ICa(L)
Some experiments were performed to investigate whether the HP used
might modulate the inhibitory effect of PK-G on basal
ICa(L). To elicit ICa(L), test pulses to
+10 mV (300 milliseconds in duration) were applied from a steady HP of
-80 mV. Leak currents were subtracted by the P/5 protocol. The
Na+-free and K+-free external solution
contained 5 µmol/L tetrodotoxin (Calbiochem-Novabiochem Co) and 30
µmol/L Ni2+ to block INa and
ICa(T). In these experiments, PK-G had a smaller
inhibitory effect on basal ICa(L) when the HP
was -80 mV than when it was -40 mV. The inhibition averaged only
8.8±1.4% (n=7, Fig 12) compared with an average value
of 42.1±2.9% (n=36) (at an HP of -40 mV).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, it was found that PK-G inhibited the basal ICa(L) as well as the stimulated ICa(L) in rat ventricular myocytes. The PK-G inhibition was variable in degree from cell to cell; large, moderate, or small degrees of inhibition were seen in all developmental stages. PK-G may directly regulate the L-type Ca2+ channels by phosphorylation of the channel protein or associated regulatory protein. The inhibition of basal ICa(L) was HP dependent: at an HP of -40 mV, the mean inhibition was 42.1% (n=36); at an HP of -80 mV, the mean inhibition was only 8.8% (n=7). Similar results have been obtained from chick embryonic heart cells, showing that intracellular application of PK-G via the patch pipette inhibited the basal and the stimulated ICa(L).16
To check whether the PK-G effect was due to its enzymatic activity, two
types of experiments were performed. When a PK-G substrate
inhibitory peptide (cG-PKI) was applied with PK-G, the
inhibitory effect on ICa(L) was blocked (Fig 5B). Heat-inactivated PK-G had little
inhibitory effect on ICa(L) (mean, 9.0±2.2%)
(Fig 5A), thus indicating that the effect exerted by PK-G resulted from
its enzymatic activity. This small inhibitory effect of
heat-inactivated PK-G may have resulted from the action
of 8Br-cGMP (0.1 µmol/L) in the test solution (mean, 10.5±1.9%;
see Fig 2).
We negated the possibility that the inhibitory action of PK-G was mediated through activation of either PDE or PPase. The possibility of involvement of the PDE was ruled out by demonstrating that the inhibitory action of PK-G was not different in the presence of a very high concentration of IBMX. That is, PK-G was still capable of depressing ICa(L) when the PDE was strongly inhibited by IBMX. The possible involvement of PPase was ruled out by demonstrating that the inhibitory action of PK-G was not different in the presence of the PPase inhibitor MC. That is, PK-G still had the ability to inhibit ICa(L) when the PPase was inhibited by MC. Therefore, the inhibitory mechanism may result from PK-G phosphorylation of the Ca2+ channel or an associated protein.
Other findings also suggest that PK-G acts via a
phosphorylation-dependent mechanism. Méry et
al9 reported that intracellular application of an active
fragment of PK-G (prepared by action of a proteolytic enzyme) inhibited
stimulated ICa(L). 8Br-cGMP inhibition of the
Ca2+-dependent slow action potentials in guinea pig cardiac
muscle was shown to occur without a decrease in cAMP
level.6 Cuppoletti et al10 have reported that
a single protein of 47 kD is specifically
phosphorylated by PK-G in guinea pig sarcolemmal
preparations.10 This protein may be a possible mediator
involved in regulation of Ca2+ channels of the heart by the
cGMP/PK-G pathway.
Recently, Haase et al25 reported that PK-A–mediated
phosphorylation of the ß-subunit of the cardiac
L-type Ca2+ channel is the major mechanism for
ß-adrenergic regulation of the channel activity. In skeletal
muscle, however, the 
1-subunit is believed to be the
relevant channel component for ß-adrenergic
regulation.26 27 Because the action of PK-G
phosphorylation is antagonistic to that of
PK-A phosphorylation, the target
phosphorylation site for PK-G must be a different site
from the site that is phosphorylated by PK-A. Such
phosphorylations by PK-A and PK-G of the
Ca2+ channel or an associated regulatory protein may
regulate the channel activity by exerting an allosteric effect.
In the present study, 8Br-cGMP (0.1 µmol/L) alone failed to
exert a significant inhibitory effect (by activating
endogenous PK-G), whereas exogenous fully activated
PK-G did (Fig 2A). We used a low subthreshold dose (0.1 µmol/L) of
8Br-cGMP as a PK-G activator. 8Br-cGMP gives strong
activation of PK-G28 but has only a weak action on PDE and
is poorly hydrolyzed (ie, long lasting). Since the concentration used
(0.1 µmol/L) was reported to give maximum activation of PK-G in
vitro,28 the PK-G applied via the patch pipette should be
maximally active. However, internal application of 8Br-cGMP (0.1
µmol/L) was almost subthreshold, whereas higher doses (1 or 10
µmol/L) significantly inhibited the basal ICa(L) (Fig 2B). These results suggest that the dose (0.1 µmol/L) used was
insufficient to activate endogenous PK-G.
Méry et al9 showed that 10 µmol/L cGMP or
8Br-cGMP was needed (internal perfusion) to give a large inhibition
(eg, 71%) of prestimulated ICa(L). Kameyama et
al3 reported that the concentration of cAMP for
half-maximal activation (internal perfusion) was 3 to 50 times
higher than that reported for in vitro studies. Hence, there is some
difference between the biochemical data and the
physiological data with respect to the
concentration of cGMP required to exert maximum effect. In summary, 0.1
µmol/L 8Br-cGMP was sufficient to maximally activate the
exogenous PK-G in the pipette but activated the
endogenous PK-G to only a small extent.
The variable effects of PK-G application on the basal ICa(L) may be explained by the working hypothesis that the level of endogenous PK-G activity may vary from cell to cell and that the basal ICa(L) is regulated by a balance of activity between PK-A and PK-G. If the endogenous PK-G activity is low, then application of exogenous activated PK-G (via the patch pipette) or addition of 8Br-cGMP into the bath should inhibit ICa(L). On the other hand, if the endogenous PK-G activity is already high, then introduction of exogenous activated PK-G should have little or no effect. This hypothesis can also explain why ISO-stimulated ICa(L) is inhibited by application of PK-G. That is, when the PK-A pathway is stimulated by ISO, the endogenous PK-G activity then becomes subordinate. Therefore, exogenous PK-G should always inhibit the stimulated ICa(L). These results are consistent with our hypothesis that PK-A and PK-G phosphorylate different target sites and that they exert antagonistic actions.
Once the applied PK-G had phosphorylated its target site, it was difficult to obtain full reversibility, even when relatively specific PK-G inhibitors were added. One possible reason for this may include the fact that the 8Br-cGMP, used as an unhydrolyzable and strong PK-G activator, causes prolonged activation of the PK-G, which results in long-lasting phosphorylation of the inhibitory regulatory subunit of the L-type Ca2+ channels. The variable effect of PK-G inhibitors on reversing the inhibition of ICa(L) can also be explained by differences in endogenous PPase activity from one cell to another. If the PPase activity is low, which may be the usual case, then the site phosphorylated by PK-G remains phosphorylated for a long time; hence, the addition of PK-G inhibitor should have little or no effect. If the PPase activity is high, then the site remains phosphorylated for only a short time and so must be rephosphorylated to remain active; therefore, the application of PK-G inhibitor should have a strong effect.
We showed that the inhibitory effect of PK-G is dependent on the HP used. So far, the reason for this is unknown. In accordance with the hypothesis described above, the endogenous PK-G activity may be generally low when the HP is -40 mV; if so, application of exogenous PK-G should exert a prominent inhibitory effect. On the contrary, when the HP is set at -80 mV, the endogenous PK-G activity may be generally high; if so, application of exogenous PK-G should give only a small inhibitory effect. HP-dependent regulation of the Ca2+ channel by PK-C activation is also reported in cultured neonatal rat ventricular cells.29 Using an HP of -80 mV, Méry et al9 reported that intracellular application of an active proteolytic fragment of PK-G gave only 5.0±3.2% inhibition of basal ICa(L) (n=4) in adult rat ventricular myocytes. The degree of inhibition is not statistically different from our finding of 8.8±1.4% inhibition (n=7) at the same HP of -80 mV. Thus, in this respect, the voltage-dependent inhibition by PK-G is similar to the inhibition produced by dihydropyridine30 and tetrodotoxin.31
Some hormones, such as ACh and ANF, are known to increase the cGMP level in mammalian cardiomyocytes.32 33 34 ACh depresses the basal ICa(L) in guinea pig atrial cells35 36 and rabbit sinoatrial nodal cells.37 38 ANF was reported to inhibit the basal ICa(L) in rat and guinea pig ventricular myocytes39 and human atrial cells.40 One possible mechanism for inhibition of the basal ICa(L) by ACh and ANF is cGMP production and the resultant PK-G phosphorylation of the Ca2+ channels or an associated protein.
In summary, in young rat ventricular myocytes, PK-G inhibited the basal ICa(L) to variable degrees (large, medium, or small), whereas PK-G always inhibited the prestimulated ICa(L) substantially back to approximately the basal level or beyond. The variable degrees of inhibition of basal ICa(L) produced by PK-G may be caused by different levels of activity of the endogenous PK-G. There were no differences in the average degree of inhibition of the basal ICa(L) during development. The inhibition of the basal ICa(L) was dependent on HP; ie, 42.1% inhibition occurred when the HP was -40 mV, whereas only 8.8% inhibition occurred when the HP was -80 mV. The inhibitory effects of PK-G were not mediated by activating PDE or PPase but most likely by a direct phosphorylation of the Ca2+ channel or an associated regulatory protein. The inhibitory effect of PK-G may be explained by a balance between activities of PK-A and PK-G in regulating the slow Ca2+ channels at two separate sites.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by National Institutes of Health grant HL-31942. We thank Prof Issei Seyama, Hiroshima University School of Medicine, for providing Dr Sumii with the opportunity to conduct joint research at the University of Cincinnati. We thank Lisa Neumeier and Sheila Blanck for preparation of the cells.
Received November 21, 1994; accepted June 15, 1995.
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References |
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