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(Circulation Research. 1995;77:803-812.)
© 1995 American Heart Association, Inc.

Articles

cGMP-Dependent Protein Kinase Regulation of the L-Type Ca²⁺ Current in Rat Ventricular Myocytes

Kotaro Sumii, Nicholas Sperelakis

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.

	Abstract

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Abstract Regulation of L-type Ca²⁺ channel current [I_Ca(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. I_Ca(L) was elicited by a depolarizing pulse to +10 mV from a holding potential of -40 mV. Stimulated I_Ca(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 I_Ca(L) was enhanced by Bay K 8644 (1 µmol/L), the enhanced basal I_Ca(L) was also reduced by PK-G. Basal I_Ca(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 I_Ca(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

	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 channel^{1 2} and in the enhancement of peak amplitude of I_Ca(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 I_Ca(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 I_Ca(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 I_Ca(L),^{9 10 11 12 13} we have evidence that cGMP does have an inhibitory effect on basal I_Ca(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 I_Ca(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 I_Ca(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 I_Ca(L). We found that PK-G inhibited both the basal I_Ca(L) as well as the prestimulated I_Ca(L).

	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 I_Ca(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 I_Ca(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 I_Ca(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 I_Ca(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 I_Ca(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 I_Ca(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 I_Ca(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:

where IC₅₀ is the dose of half-inhibition, nH is the Hill coefficient, and Max is the maximum inhibition.

The inactivation curves were fitted using the conventional Boltzmann equation:

where V_h is the potential at which half-inactivation occurs, k is the slope factor, and Max and Min are the maximum and minimum values, respectively, in the inactivation curve.

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.

	Results

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PK-G Inhibition of Prestimulated I_Ca(L)
I_Ca(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 I_Ca(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 I_Ca(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 I_Ca(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).

View larger version (23K): [in this window] [in a new window]   Figure 1. Effect of PK-G on the stimulated ICa(L) in single rat ventricular myocytes (19-day [A] and 4-day [B] rats). PK-G (50 nmol/L) was applied by using the perfusion patch-pipette technique. The PK-G was always accompanied by the unhydrolyzable PK-G activator 8Br-cGMP (0.1 µmol/L). ICa(L) was evoked by depolarizing pulses to +10 mV from an HP of -40 mV at 0.1 Hz. The pipette solution was K+ and Ca2+ free, and the bath solution was Na+ and K+ free. A, Time course of the effect of internal application of PK-G on ICa(L) prestimulated by ISO (2 µmol/L). Upper inset, The stimulatory effect of ISO (2 µmol/L), which persisted for almost 20 minutes. Lower inset, Selected current traces of ICa(L) (a, b, and c) at points denoted on the time-course curve. B, Time course of the effect of internal application of PK-G on ICa(L) prestimulated by Bay K 8644 (1 µmol/L). Upper inset, Selected current traces of ICa(L) (a, b, and c) at points denoted on the time-course curve. Lower inset, The stimulatory effect of Bay K 8644 (1 µmol/L), which persisted for almost 5 minutes.

A second method of stimulating I_Ca(L), which does not involve the cAMP/PK-A pathway, was also used. I_Ca(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 I_Ca(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 I_Ca(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 I_Ca(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 I_Ca(L) may reflect the endogenous activity of PK-A.

PK-G Inhibition of Basal I_Ca(L)
Similar experiments were performed on the basal (not stimulated through the cAMP/PK-A pathway) I_Ca(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 I_Ca(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, I_Ca(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 I_Ca(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).

View larger version (23K): [in this window] [in a new window]   Figure 2. Effect of 8Br-cGMP and PK-G on basal ICa(L) in rat ventricular myocytes. A, Time course of the effect of low doses of 8Br-cGMP (0.1 µmol/L) and PK-G (25 nmol/L) on basal (not stimulated) ICa(L) in one cell from a 4-day neonatal rat. 8Br-cGMP and PK-G were applied by using the perfusion patch-pipette technique. As shown, when 8Br-cGMP was applied in advance of the PK-G, it had only little effect, whereas subsequent addition of PK-G produced rapid inhibition. Inset, Selected current traces of ICa(L) (a, b, and c) at points denoted on the time-course curve. Experimental conditions were the same as in Fig 1. B, Dose-response relation for the inhibitory effect of internal application of 8Br-cGMP on basal ICa(L). Each data point represents the mean±SEM; numbers in parentheses indicate the number of cells for each data point. Values for IC50, the Hill coefficient, and percent inhibition (maximum) were 0.29 µmol/L, 1.32, and 52.5%, respectively.

The leak-subtracted current-voltage curves for basal I_Ca(L) before and after application of 25 nmol/L PK-G (n=11) are summarized in Fig 3A. PK-G produced a decrease in I_Ca(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 I_Ca(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 I_Ca(L) before and after application of PK-G (25 nmol/L) are illustrated in Fig 3B. As shown, PK-G decreased basal I_Ca(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 I_Ca(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.

View larger version (19K): [in this window] [in a new window]   Figure 3. The inhibitory effect of PK-G (25 nmol/L) on the current-voltage relation for basal ICa(L) in rat single ventricular myocytes (HP of -40 mV). A, Current-voltage curves of peak ICa(L) from 11 cells for the control condition () 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 -20 mV, and the voltage for peak activation occurred at +10 mV. The reversal potential shifted leftward 12 mV in presence of PK-G. *P<.05, 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 I_Ca(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.

View larger version (22K): [in this window] [in a new window]   Figure 4. Inhibitory effect of PK-G (25 nmol/L) on steady state inactivation curves of basal ICa(L) in rat single ventricular myocytes (HP of -40 mV). Data points are shown as mean±SEM (n=6). indicates the control condition; , after application of PK-G. A, Peak ICa(L) (normalized as pA/pF) plotted against various conditioning potentials. *P<.05. Inset, Voltage protocol of paired pulses. Vc and Vt indicate control and test voltages, respectively. B, Relative ICa(L) (relative to that obtained at a conditioning potential of -80 mV) plotted against conditioning potentials. Half-inactivation potential values for the control condition and after PK-G application are -22 and -28 mV, respectively. The slope factors for the control condition and after PK-G application are 5.2 and 6.5 mV, respectively.

Some experiments were performed to examine the reversibility of the PK-G inhibition of basal I_Ca(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 I_Ca(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 I_Ca(L) (data not shown).

Mechanism of Inhibition by PK-G on Basal I_Ca(L)
To check whether the effect of PK-G on basal I_Ca(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 I_Ca(L) within 5 minutes. Similar results were produced in two other experiments.

View larger version (22K): [in this window] [in a new window]   Figure 5. PK-G inhibition results from its enzymatic activity. A, Heat-inactivated PK-G showing only a slight effect on basal ICa(L) in a single ventricular myocyte from a 12-day rat. The heat-inactivated PK-G had only a slight inhibitory effect, but subsequent application of normal PK-G produced a prominent inhibition within a few minutes. Inset, Selected current traces of ICa(L) at points (a, b, and c) denoted on the time-course curve. B, Prevention of the effect of PK-G (25 nmol/L) on basal ICa(L) in a single ventricular myocyte from a 6-day rat by a PK-G inhibitor (cG-PKI, 300 µmol/L) that was applied simultaneously. After cG-PKI was washed out, the inhibitory effect of PK-G appeared. Inset, Selected current traces of ICa(L) at points (a, b, and c) denoted on the time-course curve.

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 I_Ca(L) by PK-G, and once this inhibitory peptide was removed (its perfusion stopped), the effect of PK-G to inhibit I_Ca(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 I_Ca(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 I_Ca(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 I_Ca(L) (12.0±1.2%, n=4), and internal application of PK-G (25 nmol/L) markedly inhibited I_Ca(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 I_Ca(L) (-1.6±6.7%, n=7), but internal perfusion of PK-G (25 nmol/L) inhibited I_Ca(L) (n=2, Fig 6B). In both experiments, I_Ca(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.

View larger version (24K): [in this window] [in a new window]   Figure 6. PK-G (25 nmol/L) inhibition of ICa(L) in rat single ventricular myocytes in the presence of the nonspecific PDE inhibitor IBMX (19-day [A] and 11-day [B] rats). A, Time course of the effect of internal application of PK-G on ICa(L) with 100 µmol/L IBMX. IBMX slightly increased the basal ICa(L), and PK-G greatly inhibited the current. Inset, Selected current traces of ICa(L) (a, b, c, and d) at points denoted on the time-course curve. B, Time course of the effect of internal application of PK-G on ICa(L) with 300 µmol/L IBMX. IBMX had almost no effect on basal ICa(L), and PK-G inhibited the current. Inset a, Persisting stimulatory effect of ISO (2 µmol/L) in the continued presence of IBMX (300 µmol/L), with the effect continuing after washout of the ISO. Inset b, Transient stimulatory effect of ISO (2 µmol/L) in the absence of IBMX. Note the rapid reversal of the stimulation upon washout of the ISO.

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, I_Ca(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 I_Ca(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.

View larger version (21K): [in this window] [in a new window]   Figure 7. The presence of the PPase inhibitor MC (0.5 µmol/L) did not prevent PK-G (25 nmol/L) inhibition of ICa(L). The cell illustrated was from an 8-day rat. MC was present in the patch pipette, and PK-G perfusion was started at an equilibration time of 7 minutes. Upper inset, Selected current traces of ICa(L) (a and b) at points denoted on the time-course curve. Lower inset, Plot showing the stimulatory effect of ISO. In the presence of MC (0.5 µmol/L) in the patch pipette, the stimulatory effect of ISO (2 µmol/L) continued for almost 15 minutes after washout of the ISO.

Variable Inhibitory Effect of PK-G on Basal I_Ca(L)
The average basal inhibition of I_Ca(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.

View larger version (24K): [in this window] [in a new window]   Figure 8. Variable degrees of inhibition of basal ICa(L) by PK-G (25 nmol/L) in rat single ventricular myocytes (21-day [A], 11-day [B], and 21-day [C]). PK-G (25 nmol/L) was applied using the perfusion patch-pipette technique. The effect of PK-G was large (A), moderate (B), or small (C). A through C, Time courses of the inhibitory effects of PK-G. Selected current traces at points (a and b) denoted on the time-course curve are shown on the right.

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 I_Ca(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).

View larger version (13K): [in this window] [in a new window]   Figure 9. PK-G inhibition (test potential, +10 mV) of basal ICa(L) for myocytes from rats at four different developmental stages: 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. Each data point represents the percent inhibition of basal ICa(L) produced by 25 nmol/L PK-G. Bars represent the mean±SEM for the numbers of cells given in parentheses. The average inhibitions produced at the different stages of development were not statistically significant, and variable degrees of inhibition were seen in each stage of development.

Reversibility of the Inhibited I_Ca(L) by ISO and PK-G Inhibitors
As already described, simply stopping perfusion of PK-G was insufficient to reverse the downregulated I_Ca(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 I_Ca(L) was 40.1±7.0% (n=7). As shown in Fig 10, ISO could stimulate the inhibited I_Ca(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.

View larger version (15K): [in this window] [in a new window]   Figure 10. ISO (2 µmol/L) stimulated ICa(L) that was inhibited by PK-G (25 nmol/L). Data represent a ventricular myocyte from a 6-day rat. After the basal ICa(L) was inhibited by internal application of PK-G, external application of ISO could stimulate the inhibited current. Inset, Selected current traces of ICa(L) at points (a, b, and c) denoted on the time-course curve.

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 I_Ca(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 I_Ca(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 K_i 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 I_Ca(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.

View larger version (28K): [in this window] [in a new window]   Figure 11. Variable effects of PK-G inhibitors on ICa(L) that were inhibited by PK-G in rat single ventricular myocytes (21-day [A] and 7-day [B and C] rats). A and B, Lack of reversal of the inhibition of ICa(L) produced by PK-G when PK-G inhibitors such as H-8 (5 µmol/L) and PK-G substrate peptide (cG-PKI, 300 µmol/L) were applied. C, Time course of the inhibitory effect of internal application of PK-G on basal ICa(L) and reversal of the inhibited ICa(L) to near the basal level by the PK-G inhibitor H-8. Selected current traces at points (a, b, and c) denoted on the time-course curve are shown on the right.

Voltage Dependence of the PK-G Inhibition of Basal I_Ca(L)
Some experiments were performed to investigate whether the HP used might modulate the inhibitory effect of PK-G on basal I_Ca(L). To elicit I_Ca(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 I_Na and I_Ca(T). In these experiments, PK-G had a smaller inhibitory effect on basal I_Ca(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).

View larger version (15K): [in this window] [in a new window]   Figure 12. Reduced inhibitory effect of PK-G (25 nmol/L) on basal ICa(L) when the HP was -80 mV. ICa(L) was evoked by depolarizing pulse to +10 mV from an HP of -80 mV (at 0.1 Hz). The Na+-free and K+-free external solution contained 5 µmol/L tetrodotoxin and 30 µmol/L Ni2+ to block INa and ICa(T). The time course illustrated was from a single cell from a 19-day rat. As shown, the PK-G inhibition of basal ICa(L) was small. Inset, Selected current traces of ICa(L) at points (a and b) denoted on the time-course curve.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, it was found that PK-G inhibited the basal I_Ca(L) as well as the stimulated I_Ca(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 I_Ca(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 I_Ca(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 I_Ca(L) was blocked (Fig 5B). Heat-inactivated PK-G had little inhibitory effect on I_Ca(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 I_Ca(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 I_Ca(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 I_Ca(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 I_Ca(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 I_Ca(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 I_Ca(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 I_Ca(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 I_Ca(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 I_Ca(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 I_Ca(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 I_Ca(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 I_Ca(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 I_Ca(L) in guinea pig atrial cells^{35 36} and rabbit sinoatrial nodal cells.^{37 38} ANF was reported to inhibit the basal I_Ca(L) in rat and guinea pig ventricular myocytes³⁹ and human atrial cells.⁴⁰ One possible mechanism for inhibition of the basal I_Ca(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 I_Ca(L) to variable degrees (large, medium, or small), whereas PK-G always inhibited the prestimulated I_Ca(L) substantially back to approximately the basal level or beyond. The variable degrees of inhibition of basal I_Ca(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 I_Ca(L) during development. The inhibition of the basal I_Ca(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.

	Selected Abbreviations and Acronyms

 

4-AP = 4-aminopyridine ACh = acetylcholine ANF = atrial natriuretic factor cG-PKI = PK-G peptide substrate DMSO = dimethyl sulfoxide H-8 = N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide HP = holding potential IBMX = 3-isobutyl-1-methylxanthine ICa(L) = whole-cell L-type Ca2+ current ICa(T) = T-type Ca2+ current INa = Na+ current ISO = (-)-isoproterenol hydrochloride MC = microcystin-LR PDE = phosphodiesterase PK-A = cAMP-dependent protein kinase PK-G = cGMP-dependent protein kinase PPase = protein phosphatase Rp-8-pCPT-cGMPS = 8-(4-chlorophenylthio)-guanosine-3'5'-cyclic monophosphorothioate, Rp isomer SITS = 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid

	Acknowledgments

 
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.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Reuter H. Calcium channel modulation by neurotransmitters, enzymes and drugs. Nature. 1983;301:569-574. [Medline] [Order article via Infotrieve]

2. Tsien RW, Bean BP, Hess P, Lansman JB, Nilius B, Nowycky MC. Mechanisms of calcium channel modulation by ß-adrenergic agents and dihydropyridine calcium agonists. J Mol Cell Cardiol. 1986;18:691-710. [Medline] [Order article via Infotrieve]

3. Kameyama M, Hofmann F, Trautwein W. On the mechanism of ß-adrenergic regulation of the Ca channel in the guinea-pig heart. Pflugers Arch. 1985;405:285-293. [Medline] [Order article via Infotrieve]

4. Goldberg ND, Haddox MK, Nicol SE, Glass DB, Sanford CH, Kuehl FA, Estensen R. Biologic regulation through opposing influences of cyclic GMP and cyclic AMP: the yin yang hypothesis. Adv Cyclic Nucleotide Res. 1975;5:307-330. [Medline] [Order article via Infotrieve]

5. Wahler GM, Sperelakis N. Intracellular injection of cyclic GMP depresses cardiac slow action potentials. J Cyclic Nucleotide Protein Phosphorylation Res. 1985;10:83-95. [Medline] [Order article via Infotrieve]

6. Thakkar J, Tang SB, Sperelakis N, Wahler GM. Inhibition of cardiac slow action potentials by 8-bromo-cyclic GMP occurs independent of changes in cyclic AMP levels. Can J Physiol Pharmacol. 1988;66:1092-1095. [Medline] [Order article via Infotrieve]

7. Levi RC, Alloatti G, Fischmeister R. Cyclic GMP regulates the Ca-channel current in guinea pig ventricular myocytes. Pflugers Arch. 1989;413:685-687. [Medline] [Order article via Infotrieve]

8. Wahler GM, Rusch NJ, Sperelakis N. 8-bromo-cyclic GMP inhibits the calcium channel current in embryonic chick ventricular myocytes. Can J Physiol Pharmacol. 1989;68:531-534.

9. Méry PF, Lohmann SM, Walter U, Fischmeister R. Ca²⁺ current is regulated by cyclic GMP-dependent protein kinase in mammalian cardiac myocytes. Proc Natl Acad Sci U S A. 1991;88:1197-1201. [Abstract/Free Full Text]

10. Cuppoletti J, Thakkar J, Sperelakis N, Wahler G. Cardiac sarcolemmal substrate of the cGMP-dependent protein kinase. Membr Biochem. 1989;7:135-142.

11. Hartzell HC, Fischmeister R. Opposite effects of cyclic GMP and cyclic AMP on Ca²⁺ current in single heart cells. Nature. 1986;323:273-275. [Medline] [Order article via Infotrieve]

12. Fischmeister R, Hartzell HC. Cyclic guanosine 3',5'-monophosphate regulates the calcium current in single cells from frog ventricle. J Physiol (Lond). 1987;387:453-472. [Abstract/Free Full Text]

13. Ono K, Trautwein W. Potentiation by cyclic GMP of ß-adrenergic effect on Ca²⁺ current in guinea-pig ventricular cells. J Physiol (Lond). 1991;443:387-404. [Abstract/Free Full Text]

14. Bkaily G, Sperelakis N. Injection of guanosine 5'-cyclic monophosphate into heart cells blocks calcium slow channels. Am J Physiol. 1985;248:H745-H749.

15. Tohse N, Sperelakis N. cGMP inhibits the activity of single calcium channels in embryonic chick heart cells. Circ Res. 1991;69:325-331. [Abstract/Free Full Text]

16. Haddad GE, Sperelakis N, Bkaily G. Regulation of calcium slow channels in myocardial cells by cyclic nucleotides and phosphorylation. Mol Cell Biochem. 1995;148:89-94. [Medline] [Order article via Infotrieve]

17. Conforti L, Sumii K, Sperelakis N. Diphenylamine-2-carboxylate blocks voltage-dependent Na⁺ and Ca²⁺ channels in rat ventricular cardiomyocytes. Eur J Pharmacol. 1994;259:215-218. [Medline] [Order article via Infotrieve]

18. Akita T, Joyner RW, Lu C, Kumar R, Hartzell HC. Developmental changes in modulation of calcium currents of rabbit ventricular cells by phosphodiesterase inhibitors. Circulation. 1994;90:469-478. [Abstract/Free Full Text]

19. Masuda H, Sumii K, Sperelakis N. Developmental changes in ß-adrenergic and muscarinic modulation of Ca²⁺ currents in fetal and neonatal ventricular cardiomyocytes of rat. Reprod Fertil Dev. In press.

20. Kokubun S, Reuter H. Dihydropyridine derivatives prolong the open state of Ca channels in cultured cardiac cells. Proc Natl Acad Sci U S A. 1984;81:4824-4827. [Abstract/Free Full Text]

21. Horne P, Triggle DJ, Venter JC. Nitrendipine and isoproterenol induce phosphorylation of a 42,000 dalton protein that co-migrates with the affinity labeled calcium channel regulatory subunit. Biochem Biophys Res Commun. 1994;121:890-898.

22. Frace AM, Hartzell HC. Opposite effects of phosphatase inhibitors on L-type calcium and delayed rectifier currents in frog cardiac myocytes. J Physiol (Lond). 1993;472:305-326. [Abstract/Free Full Text]

23. Colbran JL, Francis SH, Leach AB, Thomas MK, Jiang H, McAllister LM, Corbin JD. A phenylalanine in peptide substrates provides for selectivity between cGMP- and cAMP-dependent protein kinases. J Biol Chem. 1992;267:9589-9594. [Abstract/Free Full Text]

24. Hidaka H, Inagaki M, Kawamoto S, Sasaki Y. Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry. 1984;23:5036-5041. [Medline] [Order article via Infotrieve]

25. Haase H, Karezewski P, Beckert R, Krause EG. Phosphorylation of the L-type calcium channel ß subunit is involved in ß-adrenergic signal transduction in canine myocardium. FEBS Lett. 1993;335:217-222. [Medline] [Order article via Infotrieve]

26. Mundina-Weilenmann C, Chang CF, Gutierrez LM, Hosey MM. Demonstration of the phosphorylation of dihydropyridine-sensitive calcium channels in chick skeletal muscle and the resultant activation of the channels after reconstitution. J Biol Chem. 1991;266:4067-4073. [Abstract/Free Full Text]

27. Chang CF, Gutierrez LM, Mundina-Weilenmann C, Hosey MM. Dihydropyridine-sensitive calcium channels from skeletal muscle. J Biol Chem. 1991;266:16395-16400. [Abstract/Free Full Text]

28. Lincoln TM, Corbin JD. Characterization and biological role of the cGMP-dependent protein kinase. Adv Cyclic Nucleotide Res. 1983;15:140-192.

29. Lacerda AE, Rampe D, Brown AM. Effects of protein kinase C activators on cardiac Ca²⁺ channels. Nature. 1988;335:249-251. [Medline] [Order article via Infotrieve]

30. Kass RS. Ionic basis of electrical activity in the heart. In: Sperelakis N, ed. Physiology and Pathophysiology of the Heart. 3rd ed. Boston, Mass: Kluwer Academic Publishers; 1995:77-90.

31. Cohen CJ, Bean BP, Colatsky TJ, Tsien RW. Tetrodotoxin block of sodium channels in rabbit Purkinje fibers. J Gen Physiol. 1981;78:383-411. [Abstract/Free Full Text]

32. George WJ, Polson JB, O'Toole AG, Goldberg ND. Elevation of guanosine 3',5'-cyclic phosphate in rat heart after perfusion with acetylcholine. Proc Natl Acad Sci U S A. 1970;66:398-403. [Abstract/Free Full Text]

33. Cramb G, Banks R, Rugg EL, Aiton JF. Actions of atrial natriuretic peptide (ANP) on cyclic nucleotide concentrations and phosphatidylinositol turnover in ventricular myocytes. Biochem Biophys Res Commun. 1987;148:962-970. [Medline] [Order article via Infotrieve]

34. McCall D, Fried TA. Effect of atriopeptin II on Ca influx, contractile behavior and cyclic nucleotide content of cultured neonatal rat myocardial cells. J Mol Cell Cardiol. 1990;22:201-212. [Medline] [Order article via Infotrieve]

35. Iijima T, Irisawa H, Kameyama M. Membrane currents and their modification by acetylcholine in isolated single atrial cells of the guinea-pig. J Physiol (Lond). 1985;359:485-501. [Abstract/Free Full Text]

36. Cerbai E, Klöckner U, Isenberg G. Ca-antagonistic effects of adenosine in guinea pig atrial cells. Am J Physiol. 1988;255:H872-H878. [Abstract/Free Full Text]

37. DiFrancesco D, Tromba C. Muscarinic control of the hyperpolarization-activated current (i_f) in rabbit sino-atrial node myocytes. J Physiol (Lond). 1988;405:493-510. [Abstract/Free Full Text]

38. Petit-Jacques J, Bois P, Lenfant J. Mechanism of muscarinic control of the high-threshold calcium current in rabbit sino-atrial node myocytes. Pflugers Arch. 1993;423:21-27. [Medline] [Order article via Infotrieve]

39. Sorbera LA, Morad M. Atrionatriuretic peptide transforms cardiac sodium channels into calcium-conducting channels. Science. 1990;247:969-973. [Abstract/Free Full Text]

40. Le Grand B, Deroubaix E, Couétil J-P, Coraboeuf E. Effects of atrionatriuretic factor on Ca²⁺ current and Ca_i-independent transient outward K⁺ current in human atrial cells. Pflugers Arch. 1992;421:486-491.[Medline] [Order article via Infotrieve]

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				C. E. Sears, S. M. Bryant, E. A. Ashley, C. A. Lygate, S. Rakovic, H. L. Wallis, S. Neubauer, D. A. Terrar, and B. Casadei Cardiac Neuronal Nitric Oxide Synthase Isoform Regulates Myocardial Contraction and Calcium Handling Circ. Res., March 21, 2003; 92 (5): e52 - e59. [Abstract] [Full Text] [PDF]

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				J.-B. Shen and A. J. Pappano On the Role of Phosphatase in Regulation of Cardiac L-Type Calcium Current by Cyclic GMP J. Pharmacol. Exp. Ther., May 1, 2002; 301(2): 501 - 506. [Abstract] [Full Text] [PDF]

				N. Abi-Gerges, G. Szabo, A. S Otero, R. Fischmeister, and P.-F. Mery NO donors potentiate the {beta}-adrenergic stimulation of ICa,L and the muscarinic activation of IK,ACh in rat cardiac myocytes J. Physiol., April 15, 2002; 540(2): 411 - 424. [Abstract] [Full Text] [PDF]

				J. H. M. Nascimento, L. Salle, J. Hoebeke, J. Argibay, and N. Peineau cGMP-mediated inhibition of cardiac L-type Ca2+ current by a monoclonal antibody against the M2 ACh receptor Am J Physiol Cell Physiol, October 1, 2001; 281(4): C1251 - C1258. [Abstract] [Full Text] [PDF]

				M. Dittrich, J. Jurevicius, M. Georget, F. Rochais, B. K. Fleischmann, J. Hescheler, and R. Fischmeister Local response of L-type Ca2+ current to nitric oxide in frog ventricular myocytes J. Physiol., July 1, 2001; 534(1): 109 - 121. [Abstract] [Full Text] [PDF]

				G. Vandecasteele, I. Verde, C. Rucker-Martin, P. Donzeau-Gouge, and R. Fischmeister Cyclic GMP regulation of the L-type Ca2+ channel current in human atrial myocytes J. Physiol., June 1, 2001; 533(2): 329 - 340. [Abstract] [Full Text] [PDF]

				N. Abi-Gerges, R. Fischmeister, and P.-F. Mery G protein-mediated inhibitory effect of a nitric oxide donor on the L-type Ca2+ current in rat ventricular myocytes J. Physiol., February 15, 2001; 531(1): 117 - 130. [Abstract] [Full Text] [PDF]

				G. Klein, H. Drexler, and F. Schroder Protein kinase G reverses all isoproterenol induced changes of cardiac single L-type calcium channel gating Cardiovasc Res, December 1, 2000; 48(3): 367 - 374. [Abstract] [Full Text] [PDF]

				M. Nakamura, M. Sunagawa, T. Kosugi, and N. Sperelakis Actin filament disruption inhibits L-type Ca2+ channel current in cultured vascular smooth muscle cells Am J Physiol Cell Physiol, August 1, 2000; 279(2): C480 - C487. [Abstract] [Full Text] [PDF]

				K. Yuasa, H. Michibata, K. Omori, and N. Yanaka A Novel Interaction of cGMP-dependent Protein Kinase I with Troponin T J. Biol. Chem., December 24, 1999; 274(52): 37429 - 37434. [Abstract] [Full Text] [PDF]

				E. F. Du Toit, C. A. Muller, J. McCarthy, and L. H. Opie Levosimendan: Effects of a Calcium Sensitizer on Function and Arrhythmias and Cyclic Nucleotide Levels during Ischemia/Reperfusion in the Langendorff-Perfused Guinea Pig Heart J. Pharmacol. Exp. Ther., August 1, 1999; 290(2): 505 - 514. [Abstract] [Full Text]

				E. Carmeliet Cardiac Ionic Currents and Acute Ischemia: From Channels to Arrhythmias Physiol Rev, July 1, 1999; 79(3): 917 - 1017. [Abstract] [Full Text] [PDF]

				G. J. JI, B. K. FLEISCHMANN, W. BLOCH, M. FEELISCH, C. ANDRESSEN, K. ADDICKS, and J. HESCHELER Regulation of the L-type Ca2+ channel during cardiomyogenesis: switch from NO to adenylyl cyclase-mediated inhibition FASEB J, February 1, 1999; 13(2): 313 - 324. [Abstract] [Full Text]

				S. T Rapundalo Cardiac protein phosphorylation: functional and pathophysiological correlates Cardiovasc Res, June 1, 1998; 38(3): 559 - 588. [Abstract] [Full Text] [PDF]

				X. Han, I. Kubota, O. Feron, D. J. Opel, M. A. Arstall, Y.-Y. Zhao, P. Huang, M. C. Fishman, T. Michel, and R. A. Kelly Muscarinic cholinergic regulation of cardiac myocyte ICa-L is absent in mice with targeted disruption of endothelial nitric oxide synthase PNAS, May 26, 1998; 95(11): 6510 - 6515. [Abstract] [Full Text] [PDF]

				C. Depre, V. Gaussin, S. Ponchaut, Y. Fischer, J.-L. Vanoverschelde, and L. Hue Inhibition of myocardial glucose uptake by cGMP Am J Physiol Heart Circ Physiol, May 1, 1998; 274(5): H1443 - H1449. [Abstract] [Full Text] [PDF]

				N. L. Kanagy Increased vascular responsiveness to alpha 2-adrenergic stimulation during NOS inhibition-induced hypertension Am J Physiol Heart Circ Physiol, December 1, 1997; 273(6): H2756 - H2764. [Abstract] [Full Text] [PDF]

				J.-L. Balligand and P. J. Cannon Nitric Oxide Synthases and Cardiac Muscle : Autocrine and Paracrine Influences Arterioscler Thromb Vasc Biol, October 1, 1997; 17(10): 1846 - 1858. [Abstract] [Full Text]

				H. Ikenouchi, K. Kangawa, H. Matsuo, and Y. Hirata Negative Inotropic Effect of Adrenomedullin in Isolated Adult Rabbit Cardiac Ventricular Myocytes Circulation, May 6, 1997; 95(9): 2318 - 2324. [Abstract] [Full Text]

				R. Kumar, T. Namiki, and R. W. Joyner Effects of cGMP on L-type calcium current of adult and newborn rabbit ventricular cells Cardiovasc Res, March 1, 1997; 33(3): 573 - 582. [Abstract] [PDF]

				R. A. Kelly, J.-L. Balligand, and T. W. Smith Nitric Oxide and Cardiac Function Circ. Res., September 1, 1996; 79(3): 363 - 380. [Full Text]

				X. Han, L. Kobzik, J.-L. Balligand, R.A. Kelly, and T.W. Smith Nitric Oxide Synthase (NOS3)–Mediated Cholinergic Modulation of Ca2+ Current in Adult Rabbit Atrioventricular Nodal Cells Circ. Res., June 1, 1996; 78(6): 998 - 1008. [Abstract] [Full Text]

				J. W. Wegener, H. Nawrath, W. Wolfsgruber, S. Kuhbandner, C. Werner, F. Hofmann, and R. Feil cGMP-Dependent Protein Kinase I Mediates the Negative Inotropic Effect of cGMP in the Murine Myocardium Circ. Res., January 11, 2002; 90(1): 18 - 20. [Abstract] [Full Text] [PDF]

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