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Cardiovascular Research 2000 48(2):310-322; doi:10.1016/S0008-6363(00)00178-4
© 2000 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

cGMP-dependent protein kinase mediates stimulation of L-type calcium current by cGMP in rabbit atrial cells

Yang-gan Wang, Mary B. Wagner, Ronald W. Joyner and Rajiv Kumar*

The Todd Franklin Cardiac Research Laboratory, The Children's Heart Center, Department of Pediatrics, Emory University School of Medicine, 2040 Ridgewood Dr. NE, Atlanta, GA 30322, USA

* Corresponding author. Tel.: +1-404-727-5747; fax: +1-404-727-6024 rajiv{at}cellbio.emory.edu

Received 10 December 1999; accepted 26 June 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Objectives: cGMP has been shown to exert both stimulatory and inhibitory effects on cardiac L-type calcium current (ICa). The physiological role of cGMP in regulation of cardiac activity is still controversial. cGMP may be of importance in regulation of ICa in atrial cells. The present study was focused on the role of cGMP in the modulation of ICa in rabbit atrial cells. Methods: Enzymatically isolated adult rabbit atrial cells were used to measure ICa using whole cell voltage clamp. Expressed levels of cGMP-dependent protein kinase (PKG) were determined by Western blotting using PKG specific antibody in homogenates from atrial and ventricular cells. Results: Nitrosoglutathione (GSNO), a nitric oxide donor that stimulates soluble guanylyl-cyclase to elevate cGMP levels increased ICa while soluble G-cyclase inhibitors, ODQ or methylene blue inhibited ICa. Intracellular application of 8BrcGMP increased ICa and blocked the inhibitory effect of methylene blue. KT-5823, an inhibitor of PKG inhibited ICa and the stimulatory effect of GSNO was completely blocked ODQ or KT-5823. Inhibition of cAMP dependent protein kinase (PKA) by the 6–22 peptide completely blocked the stimulation of ICa by the β-agonist isoproterenol but not by GSNO. The potency of isoproterenol to stimulate ICa was very high for atrial cells (EC50 2.4±0.6 nM) and only 100 nM isoproterenol was required to stimulate ICa maximally (21.4±0.7 pA/pF) to a level (23.8±1.6 pA/pF) achieved with the inclusion of 100 µM cAMP in the pipette solution. GSNO produced an additive effect on ICa already stimulated by either 10 µM isobutylmethylxanthine (phosphodiesterase inhibitor) or a low concentration (1 nM) isoproterenol but failed to produce any effect on ICa maximally stimulated by 100 nM isoproterenol. Inhibition of PKG by KT-5823 significantly decreased the efficacy of isoproterenol and the maximal ICa achieved with 100 nM isoproterenol was decreased to 8.2±0.6 pA/pF in the presence of KT-5823. Western blot analysis showed much higher expression of PKG in atrial cells compared to ventricular cells. Conclusions: These findings suggest that stimulatory effects of cGMP on ICa in rabbit atrial cells are likely to be mediated via PKG dependent phosphorylation of calcium channels or associated proteins and that the effects of cGMP are not antagonistic to cAMP. PKG is highly expressed in atrial cells and PKG dependent phosphorylation may be necessary for maintaining basal ICa and fully stimulating ICa by β-adrenergic activation in atrial cells.

KEYWORDS Second messengers; Protein kinases; Nitric oxide; Ca-channel; Myocytes; Atrial function


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Regulation of ICa is a complex interaction among many factors, including circulating hormones, localized release of neurotransmitters, intracellular release and accumulation of calcium, and the effects of many pharmacological agents. The heart is a complex organ with many regional variations in cell membrane properties, cell shape and orientation, and cellular electrical coupling. Nevertheless, all of the regions of the heart are simultaneously exposed to circulating hormones as well as drugs which are used for treatment of the symptoms of cardiac dysfunction even though the dysfunction may not be uniformly present in different cardiac regions. We recently demonstrated the importance of basal values of ICa and the effects of modulating ICa on the ability of cells to propagate action potentials [9]. There has been increasing interest in the action potential duration and refractoriness of atrial cells as important components of the maintenance of atrial fibrillation [3] and the L-type calcium current (ICa) has been shown to be modified as a consequence of atrial fibrillation [18,31,32].

Various studies on the effects of cGMP on the L-type calcium current (ICa) in different species have failed to observe consistent effects of cGMP. Most of the previous studies on effects of cGMP on ICa were focused on ventricular cells and it was generally shown that cGMP exerts opposite contractile and electrical responses in the heart to those elicited by cAMP. Both inhibitory [8,17,28] and stimulatory [21] effects of cGMP on ICa in ventricular myocytes have been observed, especially in the presence of elevated cAMP levels. cGMP activates a specific cGMP dependent protein kinase (PKG) which can phosphorylate a number of proteins and can also stimulate or inhibit specific phospodiesterases (PDEs) to produce either antagonistic or synergistic effects with cAMP elevating agents. Three pathways for the modulation of ICa by cGMP have been demonstrated: (i) activation of an endogenous PKG, which leads to the stimulation of ICa in newborn [16] or juvenile [6] but not adult [16] rabbit ventricular cells and leads to inhibition of ICa in guinea pig [17,21] or rat [19] ventricular cells; (ii) activation of cGMP-stimulated PDE that leads to the inhibition of ICa in frog ventricular myocytes [8] and human atrial myocytes [24]; and (iii) inhibition of cGMP inhibited PDE which results in an increase of ICa in human atrial cells [11] and guinea pig ventricular cells [21].

Most of the previous studies have shown no significant effect of cGMP on basal ICa, i.e. in the absence of elevated cAMP. However, we have recently shown [16] that elevation of intracellular cGMP level either by internal perfusion of cGMP analogs or by stimulation of guanylyl cyclase (G-cyclase) by NO donors, significantly stimulates basal ICa in newborn rabbit ventricular cells but not in adult rabbit ventricular cells. We have also shown that the basal levels of cGMP in isolated ventricular myocytes were significantly higher in newborn compared to adult heart [14]. Our recent work on cDNA cloning and expression of PKG in rabbit heart cells [15] has clearly shown that PKG mRNA and protein levels were much greater in newborn rabbit ventricular cells compared to adult rabbit ventricular cells. In contrast to our studies on adult rabbit ventricular cells, Han et al. [7] have shown PKG-dependent stimulation of basal ICa, while Tohse et al. [26] have shown a small reduction (15–20%) in basal IBa by atrial natriuretic peptide and by extracellular application of 300 µM 8BrcGMP. These findings suggested that cGMP may be physiologically important for the regulation of ICa in newborn rabbit ventricular cells but not in adult rabbit ventricular cells.

Compared to ventricular cells, not much work has been done on atrial cells and very little is known about the role of cGMP on ICa in atrial cells. The work by Fischmeister and colleagues [11,24] on human atrial cells has shown both stimulatory and inhibitory effects of cGMP. To gain more insights into the possible role of cGMP in the regulation of ICa in atrial cells, we studied the regulatory role of cGMP on ICa in isolated adult rabbit atrial myocytes. In the present study, we examined the effects of cGMP on ICa by elevating cGMP levels (either by intracellular application of cGMP analogs or by stimulating G-cyclase by a NO donor) and by lowering cGMP levels (by G-cyclase inhibitors methylene blue or ODQ). We have also examined the response of elevated cGMP levels in the presence of inhibitors of PDEs, PKA or PKG to elucidate the mechanism of cGMP action on ICa in rabbit atrial cells. To study the interaction between PKA and PKG mediated pathways on ICa, we initially examined the effects of β-agonist isoproterenol and the intracellular application of cAMP to elucidate cAMP mediated regulation of ICa in rabbit atrial cells and then we tested the response of isoproterenol under the conditions of PKG inhibition by KT-5823. We also analyzed specific expression of PKG protein in homogenates of atrial and ventricular cells to determine the significance of PKG levels in the regulation of cardiac calcium channels.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 Acknowledgments
 References
 
2.1 Preparation of isolated cells
Single atrial and ventricular myocytes were prepared from adult New Zealand White rabbits weighing 2.5–3.5 kg as we described earlier [5,13]. The rabbits were heparinized with 500 U heparin i.v., and anesthetized using 50 mg/kg pentobarbital. The heart was rapidly removed via thoracotomy with artificial respiration, and the aorta was cannulated for Langendorff perfusion. For atrial cells, the cannulated heart was perfused sequentially at 37°C with the base solution containing 0.9 mM CaCl2 for 4 min., the base solution containing 50 µM EGTA for 5 min., and the base solution containing 100 µM CaCl2, 1 mg/ml collagenase (Worthington-type IIA), and 0.07 mg/ml protease (Sigma-type XIV) for 6 min at 36–37°C. After enzyme perfusion, the interatrial septum was excised, cut into thin strips, and further digested in the recirculated enzyme solution used above for 5–8 min at 36–37°C with continuous stirring. Cells were isolated by triturating the tissue strips in storage solution and were then placed at 4°C in storage solution. Single ventricular cells were isolated as we described earlier [13]. Isolated cells were stored at 4°C.

2.2 Recording of whole cell calcium current
The cells were placed in a chamber that was continuously perfused with normal Tyrode's solution at 2 ml/min at room temperature. The cells that were quiescent and had a rod shaped appearance were used in this study. Voltage-clamp experiments were performed as we described earlier [22] in the whole cell configuration of the patch-clamp method by use of an Axopatch-200 patch-clamp amplifier (Axon Instruments, Foster City, CA, USA). For routine monitoring of ICa, the cells were depolarized every 10 s from a holding potential of –45 mV to a test potential of 0 mV for 360 ms. The test potential for atrial cells is based on the peak current of the I–V relationships for both control and isoproterenol stimulated ICa (data not shown). The elicited ICa was filtered at a corner frequency of 2 kHz, digitized at 200-µs intervals, and stored and analyzed on a Pentium computer using PCLAMP6 software (Axon Instruments). ICa was measured as the peak inward current. Membrane capacitance was measured using the calibrated capacity compensation circuit of the Axopatch voltage clamp amplifier using 5 mV hyperpolarizing pulses. We expressed all the current data as current density (pA/pF) by normalizing the peak calcium current for each cell to the cell capacitance. Cells (10–20%) which showed rundown were excluded from the analysis by only accepting the data from cells in which ICa was stable in the control condition and reached a steady state with the tested drugs.

2.3 Solutions and drugs
Base solution (in mM): 130 NaCl, 4.5 KCl, 3.5 MgCl2, 0.4 NaH2PO4, 5-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and 10 dextrose, pH 7.25. Normal tyrode (in mM): 148.8 NaCl, 4 KCl, 1.8 CaCl2, 0.53 MgCl2, 0.33 NaH2PO4, 5 HEPES, and 5 dextrose, pH 7.4. Ca2+-free solution (in mM): 148.8 NaCl, 4 KCl, 0.53 MgCl2, 0.33 NaH2PO4, 5 HEPES, and 5 dextrose, pH 7.4. Storage solution (in mM): 100 K-glutamate, 25 KCl, 10 KH2PO4, 0.5 ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1 MgSO4, 20 taurine, 5 HEPES, and 10 dextrose, pH 7.2. ICa test solution (in mM): 130 NaCl, 1.8 CaCl2, 20 CsCl, 0.53 MgCl2, 5 HEPES, 5 glucose, pH 7.4. Normal pipette solution (in mM): 110 CsOH, 90 aspartic acid, 20 CsCl, 10 tetraethylammonium Cl (TEACl), 5 HEPES, 10 EGTA, 5 MgATP, 5 Na2 creatine phosphate, 0.4 GTP (Tris), 0.1 leupeptin, pH 7.2. Modifications to these solutions are described below as appropriate for specific protocols. Methylene blue, 8BrcGMP, isoproterenol, IBMX and GSNO were obtained from Sigma (St. Louis, MO, USA), KT-5823 and PKA inhibitor (PKI, 6–22) from Calbiochem (La Jolla, CA, USA) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) from Biomol (Plymouth Meeting, PA, USA). KT-5823, ODQ, and IBMX were dissolved in DMSO. Isoproterenol stock solution was made in 1 mM ascorbic acid. GSNO solution was made fresh in light-protected containers. All other agents were dissolved in deionized water.

2.4 Preparation of total homogenates from isolated cells
The total homogenates from isolated adult rabbit atrial and ventricular cells were prepared by a method we described earlier [15]. Briefly, isolated cells were homogenized in hypotonic membrane buffer (containing in mM, Tris–HCl (pH 7.5) 50, MgCl2 5, EDTA 1, dithiothreitol 1, Pepstatin A 0.001, phenylmethylsulfonyl fluoride 0.4, phenanthroline 1 and iodoacetamide 1) by sonication. The homogenate was then centrifuged at 100 g for 5 min to separate the unbroken cells, and the supernatant (total homogenate) was stored at –70°C in small aliquots. Protein was determined by a dye method (Bio-Rad, Hercules, CA, USA).

2.5 Western blot analysis
Immunological characterization and quantification of the amounts of PKG type I in homogenates prepared from enzymatically dissociated atrial and ventricular cells was performed by using a method we described [15] recently for determining PKG expression in adult and newborn rabbit ventricular cells. A polyclonal antibody that recognizes specifically PKG type I was kindly provided by Dr. Thomas M. Lincoln, University of Alabama (Birmingham, AL, USA). After electrophoretic transfer of proteins on PVDF membrane (Amersham, Arlington Heights, IL, USA), membranes were blocked (TBS containing 0.1% Tween 20 and 5% nonfat dry milk) and then incubated overnight with an affinity-purified PKG antibody at 1:1000 dilution in blocker solution at 4°C. Following several washes in wash buffer (TBS containing 0.05% SDS, 0.05% NP-40 and 0.125% sodium deoxycholate), the membranes were incubated with horseradish peroxidase conjugated secondary antibody diluted at 1:10 000 in the blocker solution for 1 h at room temperature. For the detection of bands, we used enhanced chemiluminescence detection (ECL+plus, Amersham, Arlington Heights, IL, USA) using Lumigen PS-3. To compare the relative amounts of PKG in atrial versus ventricular preparations, we use a two dimensional gel imaging system (ALPHA IMAGER 2000, documentation and analysis system, Alpha Innotech, USA) with multiple atrial and ventricular preparations and purified PKG as positive control run on different tracks of the same gel. Peak area obtained for atrial and ventricular bands was normalized using peak area for positive control to get the relative amount of PKG present per mg protein of isolated cells.

2.6 Statistics
All values are presented as mean±SEM. Statistical analysis was performed using SIGMASTAT for Windows with paired or unpaired t-test. A P value of less than 0.05 was defined as significant.

The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 Acknowledgments
 References
 
3.1 Stimulating guanylyl cyclase increases basal ICa
We have shown previously [16] that activation of soluble G-cyclase by the NO donor nitrosoglutathione (GSNO) increases basal ICa in newborn rabbit ventricular cells. In order to investigate the effect of increasing intracellular cGMP levels on basal ICa of rabbit atrial cells, we applied GSNO in the extracellular perfusion solution. Fig. 1 shows the stimulatory effect of GSNO on basal ICa for a rabbit atrial cell. GSNO significantly increased basal ICa in a concentration dependent manner. Since the calcium current is an inward current, the current traces and time course of ICa in this and other Figures are shown as negative currents. However, throughout the text and in other Figures, we refer to the magnitude of calcium current density as positive values representing the average of normalized peak calcium current density. For this cell, the basal ICa was increased from 4.8 pA/pF (trace labeled 1 in part B) to 6.3 pA/pF (trace labeled 2 in part B) and then to 9 pA/pF (trace labeled 3 in part B) by 10 µM and 100 µM GSNO, respectively. On washout of GSNO, ICa reversed back to the control values. The average increase of ICa by external application of 10 µM and 100 µM GSNO was 30.5% (from 7.4±0.2 to 9.6±0.5 pA/pF, n = 4, P<0.05) and 69.8% (from 6.6±0.7 to 11.1±1.2 pA/pF, n = 5, P<0.05), respectively. To rule out the possible stimulation of ICa by reduced glutathione (GSH) that is produced by cleavage of GSNO, we tested three cells by applying 30 µM GSH in the extracellular solution with no effect on ICa (data not shown).


Figure 1
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  		Fig. 1 (A) Time course of ICa (pA/pF) for an atrial cell with exposure first to 10 µM and then to 100 µM nitrosoglutathione (GSNO) in the external solution. The time period of exposure is indicated by the horizontal arrows. To wash GSNO, cell was exposed to normal test solution. (B) The superimposed ICa current recordings where current traces labeled as 1, 2 and 3 were obtained at the times marked by the corresponding letters in (A). The labels 1, 2, and 3 represent the steady-state ICa level at the period of control and the application of 10 µM and 100 µM GSNO, respectively.

 
3.2 Blocking guanylyl cyclase decreases basal ICa and prevents effects of GSNO
ODQ and methylene blue (MB) are soluble G-cyclase inhibitors commonly used to decrease cGMP levels. In order to test the effect of lowered cGMP levels on basal ICa, we used ODQ or MB to lower intracellular cGMP levels. Fig. 2A summarizes the effects on five cells of 10 µM ODQ alone and also the effects for the same cells of the addition of 100 µM GSNO in the continuing presence of 10 µM ODQ on basal ICa in rabbit atrial cells. A 10-µM ODQ solution produced a significant inhibition of basal ICa by 25% (from 6.8±1.1 to 5.1±0.6 pA/pF, n = 5, P<0.05). To confirm whether the inhibitory effect of ODQ on ICa is mediated by lowering of cGMP levels due to the inhibition of soluble G-cyclase, we used 100 µM GSNO in the presence of ODQ. The presence of ODQ completely blocked the stimulatory response of GSNO on ICa. The average ICa in the presence of 10 µM ODQ alone was not significantly altered by adding 100 µM GSNO to the extracellular solution (5.0±0.6, n = 5, NS).


Figure 2
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  		Fig. 2 (A) Bar graph showing average ICa densities in control solution, with extracellular application of 10 µM ODQ, and with extracellular application of 100 µM GSNO in the presence of 10 µM ODQ for atrial cells. (B) Bar graph showing average ICa densities in control solution, with extracellular application of 10 µM methylene blue (MB), with intracellular application of 30 µM 8BrcGMP, and with 10 µM MB in the presence of internally applied 8BrcGMP. Vertical bars indicates standard error of mean of the number of experiments indicated near the bars; *, significantly different compared to control; **, significant difference between the two groups; NS, values are not significantly different.

 
3.3 Increasing intracellular cGMP increases ICa and prevents the decrease in ICa produced by blocking guanylyl cyclase
To further evaluate the effects of altered levels of cGMP on ICa, we used 8-bromo-cGMP (8BrcGMP, a nonhydrolyzable analog of cGMP) in the pipette solution to increase cGMP levels and tested its effects with and without a G-cyclase inhibitor, MB. Fig. 2B summarizes the effects of external application of 10 µM MB for cells with a normal pipette solution, of internal application of 30 µM 8BrcGMP, and of the external application of 10 µM MB with continued internal presence of 30 µM 8BrcGMP on ICa for rabbit atrial cells. With a normal pipette solution, the application of 10 µM MB produced a significant inhibition of basal ICa by 33.4±6% (n = 5, P<0.05). The presence of 8BrcGMP (30 µM) in the pipette solution significantly increased basal ICa by 128% from the control level (6.3±0.3 pA/pF, n = 36, determined from cells without 8BrcGMP in the pipette solution) to 14.4±1.5 pA/pF (n = 11, P<0.05). To examine whether the inhibitory effect of MB on ICa is mediated by lowering the cGMP levels, we applied 10 µM MB to the bath solution in the continuing presence of 30 µM 8BrcGMP in the pipette solution. The excess amount of 8BrcGMP inside the cell completely blocked the inhibitory effect of MB on ICa.

3.4 Blocking PKG decreases basal ICa and prevents positive effects of GSNO while blocking PDEs increases ICa and is additive to the GSNO effects
To determine whether the stimulatory effect of cGMP on basal ICa is through its action on PKG or through its indirect action on cAMP dependent protein kinase (PKA) via cGMP dependent phosphodiesterases, we further used specific inhibitors to block two different pathways. To evaluate the involvement of PKG in the maintenance of basal ICa, we used 0.6 µM KT-5823 to inhibit PKG. These data are summarized in Fig. 3 as percentages of resulting ICa with respect to the control ICa (100%). Fig. 3 summarizes the effects of 100 µM GSNO applied alone, 0.6 µM KT-5823 applied alone, and the application of 100 µM GSNO in the continuing presence of KT-5823. The average basal ICa was increased by 77% (177±25% of control) by 100 µM GSNO alone and was decreased by 42% by 0.6 µM KT-5823 (from 7.3±1.0 pA/pF to 4.2±0.4 pA/pF, n = 5, P<0.05). The effect of 100 µM GSNO in the continuing presence of 0.6 µM KT-5823 was completely blocked (the average ICa was 4.0±0.2 pA/pF, n = 5, NS compared to ICa with KT-5823 alone). In similar experiments performed on ten isolated rabbit ventricular cells, application of 0.6 µM KT-5823 had no significant effect on basal ICa (control ICa 5.1±0.3 vs. 5.0±0.2 pA/pF with 0.6 µM KT-5823).


Figure 3
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  		Fig. 3 Bar graph showing mean values of percentage of control ICa (taken as 100%) with the treatment of 100 µM GSNO alone, 0.6 µM KT-5823 alone, 100 µM GSNO in the presence of 0.6 µM KT-5823, 10 µM IBMX alone, and 100 µM GSNO in the presence of 10 µM IBMX. Vertical bars indicate standard error of mean of the number of experiments indicated near the bars; *, significantly different compared to control; **, significant difference between the two groups; NS, values are not significantly different.

 
It is known that cGMP can also produce its effect on ICa by inhibiting or stimulating specific cGMP sensitive phosphodiesterases (PDEs). To explore the possible involvement of PDEs in the stimulatory effect of cGMP, we used IBMX, a nonspecific PDE inhibitor at a concentration able to produce a more than 100% increase in ICa. Fig. 3 also summarizes the effects on ICa of 10 µM IBMX alone and of 100 µM GSNO added in the continuing presence of 10 µM IBMX. 10 µM IBMX alone in the bath solution increased basal ICa by 139% (239±19% of control, from 4.4±0.4 to 10.1±0.7 pA/pF, n = 8, P<0.05). The addition of 100 µM GSNO in the continuing presence of 10 µM IBMX produced an additional increase over IBMX alone to an overall increase of 201% (301±27% of control, the average ICa increased to 12.8±1.0 pA/pF, n = 8, P<0.05). This indicates that the stimulatory effect of GSNO is not mediated by PDEs since ICa stimulation by GSNO was not blocked by the inhibition of PDEs. The above results suggested that changes in cGMP levels in atrial cells play a crucial role in determining basal ICa and that the stimulatory effect of cGMP are possibly mediated through PKG.

3.5 PKA inhibition blocks the stimulation of ICaby isoproterenol, but not by GSNO
To further confirm that PKA does not mediate the stimulatory effect of cGMP, we examined the effect of GSNO under conditions in which PKA had been inhibited and the stimulatory effect of isoproterenol was almost completely blocked. Fig. 4 shows the results from a rabbit atrial cell for which we included 2 µM PKI in the pipette solution to inhibit PKA. The presence of PKI produced a gradual decline in basal ICa after dialysis of the pipette solution started. After reaching a nearly stable level (4.2 pA/pF) in the presence of PKI, we then applied 10 nM isoproterenol in the bath solution. The presence of PKI in the pipette solution completely blocked the stimulatory effect of 10 nM isoproterenol and ICa further declined slowly to 3.8 pA/pF. We then applied 100 µM GSNO in the continuing presence of 10 nM isoproterenol and obtained a rapid increase of ICa to 5.6 pA/pF, an increase of 47% from the value prior to the application of GSNO. For six cells, the intracellular application of 2 µM PKI produced an average ICa of 4.8±0.6 pA/pF and this was slightly decreased to 4.6±0.7 (P = 0.4) in the presence of 10 nM isoproterenol. The application of 100 µM GSNO in the continued presence of PKI and isoproterenol increased ICa to 7.3±0.8 pA/pF (P<0.05), a percentage increase (59%) very close to the percentage increase elicited by 100 µM GSNO in the absence of PKI (77%). These results indicate that the stimulatory effect of cGMP was mediated by PKG and not by PKA.


Figure 4
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  		Fig. 4 (A) Time course of ICa (pA/pF) for an atrial cell with intracellular exposure to 2 µM PKI (6–22 peptide included in the internal pipette solution to inhibit PKA). The cell was first superfused with normal external solution, then with 10 nM isoproterenol alone and finally with 100 µM GSNO+10 nM isoproterenol. The time period of exposure is indicated by the horizontal arrows. (B) The superimposed ICa current recordings where current traces labeled as 1, 2 and 3 were obtained at the times marked by the corresponding letters in (A). The labels 1, 2, and 3 represent the steady-state ICa level in control, 10 nM isoproterenol, and 100 µM GSNO+10 nM isoproterenol, all recorded in the presence of PKI in the pipette solution, respectively.

 
3.6 Effect of isoproterenol on Ica in atrial cells
The increase in ICa by β-adrenergic stimulation was examined quantitatively with various concentrations of isoproterenol. We used five atrial cells to test the effect of increasing concentrations of isoproterenol on the same cell. Fig. 5 shows superimposed current recordings and the time course of peak ICa increase (inset) by increasing concentrations of isoproterenol recorded from an isolated rabbit atrial cell. The ICa in control external solution has peak amplitude of 6.1 pA/pF. ICa started to increase on applying 0.1 nM isoproterenol and the response to 0.1 nM isoproterenol is an increase of ICa by 31% (to 8 pA/pF, peak B), indicating a high potency for isoproterenol. ICa was further increased with increasing doses of isoproterenol and finally reached a maximum (23.8 pA/pF, an increase by 290%) at 100 nM isoproterenol (peak E). ICa density returned to the control values on subsequently washing out isoproterenol (data not shown).


Figure 5
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  		Fig. 5 Calcium current (ICa) recordings before and after treatments with varying concentrations of isoproterenol from an atrial cell. Ordinate represents ICa density (pA/pF) obtained by normalizing the current to the membrane capacitance. The current traces were recorded in control conditions, and in the presence of 0.1, 1, 10, and 100 nM isoproterenol. The inset shows the time course of peak ICa as the several concentrations of isoproterenol are successively applied.

 
3.7 Inhibition of PKG decreases the efficacy of isoproterenol to increase ICa in rabbit atrial cells
The physiological role of cGMP in regulation of cardiac activity is generally considered antagonistic to cAMP [4]. To gain insight into the interaction of intracellular cGMP and cAMP on the regulation of ICa, we further investigated the interaction of PKG and PKA on ICa. The two pathways may have significant interactions if the two kinases act on either the same or different phosphorylation sites of the calcium channel. To examine the interactions between PKA and PKG mediated regulation of ICa, we investigated the effects of PKG inhibition on stimulation of ICa by isoproterenol. For this, we compared the ability of KT-5823 to inhibit ICa stimulated by various concentrations of isoproterenol in atrial cells. Fig. 6A shows the time course of peak ICa for a rabbit atrial cell before and after application of KT-5823 (0.6 µM) and then the application of 100 nM isoproterenol plus 0.6 µM KT-5823 after reaching the steady state of ICa with KT-5823. ICa decreased from 5.8 to 3.3 pA/pF (43%) after the application of KT-5823. On applying isoproterenol (100 nM) in the continuing presence of 0.6 µM KT-5823, ICa increased to 8.4 pA/pF (154% increase over ICa inhibited by KT-5823). Fig. 6B shows the effect of 100 nM isoproterenol alone for a separate atrial cell from the same heart for comparison. For this cell, ICa increased from 4.8 to 15 pA/pF (an increase of 213% over control) by 100 nM isoproterenol. On comparing the effects of isoproterenol with and without KT-5823 (132±17%, n = 4 and 243±11%, n = 7, respectively), it is clear that KT-5823 reduced the response to isoproterenol of ICa.


Figure 6
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  		Fig. 6 (A) Time course of ICa (pA/pF) for an atrial cell with control external solution, then with the application of 0.6 µM KT-5823 to inhibit PKG, and then the addition of 100 nM isoproterenol to the external solution. (B) Time course of ICa (pA/pF) for a different atrial cell (from the same heart) with normal external solution, then with 100 nM isoproterenol. The exposure times are indicated by the horizontal arrows.

 
Fig. 7 summarizes the dose dependent effects of isoproterenol on ICa density (A) and the percentage increase in ICa (B) in the absence and presence of 0.6 µM KT-5823. Isoproterenol alone caused a dose dependent increase in the ICa density with an apparent threshold concentration of 0.1 nM. The maximal ICa density (21.4±0.7 pA/pF, n = 5) was produced by 100 nM isoproterenol. Effect of intracellular application of cAMP is shown by the horizontal continuous line. For five cells, we included 100 µM cAMP in the pipette solution and this produced an ICa level of 23.8±1.6 pA/pF (n = 5) that was equivalent to the maximal current density obtained in the presence of isoproterenol. The concentration of isoproterenol for one half of the maximal effect (EC50) was 2.4±0.6 nM. This showed that the potency of isoproterenol to stimulate ICa was much higher for atrial cells compared to rabbit ventricular cells (51±8 nM [23]). KT-5823 (0.6 µM) produced about 45% inhibition of basal ICa and for 17 cells used, the average ICa decreased from 6.4±0.3 to 3.5±0.1 pA/pF. KT-5823 caused a significant reduction of response to isoproterenol at all concentrations tested (1 nM to 1 µM). The maximal ICa achieved with 100 nM isoproterenol was substantially decreased from 21.4±0.7 (n = 7) to 8.2±0.6 pA/pF (n = 4, P<0.05) in the presence of KT-5823. Since the addition of 0.6 µM KT-5823 significantly reduced the basal ICa, we have replotted the dose–response relationship of Fig. 7A as percent increase in ICa produced by isoproterenol in Fig. 7B with all results normalized to the values obtained either in the control solution or in the 0.6 µM KT-5823 solution just prior to the addition of the isoproterenol. The maximum percentage increase in ICa by 100 nM isoproterenol alone (243±11%, n = 7) was decreased to 132±17% (n = 4) in the presence of KT-5823. This clearly indicates that PKG dependent basal phosphorylation may be necessary for maximal stimulation of ICa by isoproterenol in atrial cells.


Figure 7
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  		Fig. 7 Graphs showing the dose response curves of the effect of isoproterenol on ICa in the absence (filled circles) and presence (open squares) of 0.6 µM KT-5823 for atrial cells. (A) ICa density (pA/pF) obtained by normalizing the current to the membrane capacitance. (B) Percent increase in ICa by varying doses of isoproterenol over values obtained in the control solution or in the KT-5823 solution just prior to the application of the isoproterenol. Data points show the mean values of the number of cells indicated near the symbol, vertical bars represent the S.E.M. The horizontal dashed line in (A) shows the average maximum ICa achieved for five cells for which 100 µM cAMP was included in the patch pipette solution. Data points show the mean values of the number of cells indicated near the symbol, vertical bars represent standard error of mean. *, Significant difference comparing data without KT-5823 to that obtained for the same isoproterenol dose with KT-5823.

 
3.8 With low isoproterenol doses, GSNO still increases ICa, but with high isoproterenol doses the effect of GSNO is abolished
We further tested the interactions between cAMP and cGMP mediated pathways by investigating the effects of increased cGMP levels in the presence of a low (1 nM) or maximal concentration (100 nM) of isoproterenol to stimulate ICa. Fig. 8 summarizes the effects of 1 nM or 100 nM isoproterenol alone, the effects of 100 µM GSNO in the continued presence of the 1 nM or 100 nM isoproterenol, and the effect of 1 nM isoproterenol in the continued presence of intracellular 8BrcGMP (30 µM) with the data plotted as percent increase in ICa. For four cells the average effect of 1 nM isoproterenol was to raise ICa by 71±8% (from 6.2±0.6 to 10.9±0.9 pA/pF, P<0.05). With the continued presence of 1 nM isoproterenol, 100 µM GSNO further increased ICa to 12.4±0.8 pA/pF (107±8%, P<0.05). GSNO produced a statistically significant change in ICa from the value obtained with 1 nM isoproterenol alone. When we exposed cells to 100 nM isoproterenol, ICa increased from 7.0±1.0 to 21.7±1.8 pA/pF (220±22%, n = 4). In the continued presence of 100 nM isoproterenol, we then further exposed the cells to 100 µM GSNO with no significant change in ICa (21.5±2.1 pA/pF, n = 4, P>0.05). We also studied three cells in which we included 30 µM 8BrcGMP in the pipette solution, producing an average ICa of 12.2±1.1 pA/pF (94±17% increase over control) and then applied 1 nM isoproterenol in the continuing presence of 30 µM 8BrcGMP, producing an average ICa of 21.7±2.3 pA/pF (244±36% increase over control). The combined effect of 30 µM 8BrcGMP in the pipette solution and 1 nM isoproterenol on ICa (21.7±2.3 pA/pF) was much greater (P<0.05) than the effect produced by 1 nM isoproterenol (10.9±0.9 pA/pF) with a control pipette solution.


Figure 8
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  		Fig. 8 Bar graph showing mean values of percent increase in ICa in three groups of cells. The left two bars show the values for 1 nM isoproterenol alone, and for 100 µM GSNO in the presence of 1 nM isoproterenol. The middle two bars show the values for intracellular 30 µM 8BrcGMP and for 1 nM isoproterenol in the continued presence of 30 µM 8BrcGMP. The right two bars show the values with 100 nM isoproterenol alone and for 100 µM GSNO in the presence of 100 nM isoproterenol. Vertical bars show S.E.M. of the number of experiments indicated near the bars; *, significant difference comparing the effect of isoproterenol versus the effect of isoproterenol plus GSNO; **, significant difference comparing the combined effect of 1 nM isoproterenol and 30 µM 8BrcGMP versus the effect of 1 nM isoproterenol alone. NS, values are not significantly different.

 
3.9 Expressed levels of PKG are much greater in atrial than in ventricular cells
One possible reason for the presence of regulation of ICa by PKG in rabbit atrial cells, even though this regulation seems to be absent in adult rabbit ventricular cells, would be that atrial cells might have significantly greater expression of PKG than ventricular cells. We assessed the levels of PKG protein by Western blot analysis in homogenates prepared from isolated atrial and ventricular myocytes of adult rabbit heart using a PKG type-1 specific antibody. Fig. 9 shows a representative immunoblot in which we loaded 4 ng of purified bovine lung PKG type I{alpha} in the central lane (labeled PKG) and included in separate lanes 50 µg of homogenate protein prepared from isolated myocytes of three different atrial (labeled AT) and three different ventricular (labeled VT) preparations. The results indicate an immunoreactive protein at an apparent molecular weight of 79 kDa in atrial preparations that was aligned with the positive control of PKG type I{alpha}, with no visible bands at the corresponding locations for the ventricular samples. The PKG type I levels were much higher in homogenates from atrial cells (n = 6) compared to ventricular cells (n = 6), from which PKG levels were too low to be detected. We quantified the relative amount of PKG type I in atrial and ventricular cells by normalizing the atrial and ventricular band density to PKG positive control. The relative amount of PKG type I present in atrial cells was (61.9±10.1 ng/mg protein, n = 6).


Figure 9
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  		Fig. 9 Immunoblot analysis of PKG in total homogenates prepared from isolated atrial and ventricular cells of adult rabbit hearts. The three lanes marked as AT (atrial) and VT (ventricular) contained 50 µg homogenate protein from atrial and ventricular cells respectively. The replicate lanes represent samples from different animals. The lane marked as PKG contained 4 ng purified bovine lung PKG.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
Acknowledgments
 References
 
cGMP has been considered as a generally inhibitory modulator of cardiac function. However, both positive and negative regulatory effects of intracellular cGMP on ICa in cardiac myocytes have been reported and attributed to several different mechanisms. The present study was focused on the modulation of L-type calcium current by cGMP and its mechanism of action in rabbit atrial cells. The salient findings of this study are summarized as follows:

1. The soluble G-cyclase stimulator GSNO increased basal ICa, while soluble G-cyclase inhibitors ODQ or methylene blue and PKG inhibitor KT-5823 lowered basal ICa. The stimulatory effect of GSNO was completely blocked by either ODQ or KT-5823.
2. Intracellular application of 8BrcGMP (a more specific PKG stimulant than native cGMP) significantly increased basal ICa and prevented the inhibitory effect of methylene blue.
3. Inhibition of PKA by PKA inhibitor peptide blocked the stimulatory effect of isoproterenol on ICa but failed to block the stimulatory effect of GSNO.
4. Inhibition of PDEs by IBMX did not block the stimulatory effect of GSNO and GSNO produced an additive effect on ICa already stimulated by IBMX.
5. The potency of isoproterenol to stimulate ICa was much greater for atrial cells compared to ventricular cells.
6. The stimulatory effect of GSNO was present under intermediate stimulation of ICa by isoproterenol but blocked after maximal stimulation of ICa by a saturating dose of isoproterenol.
7. Inhibition of PKG decreased the efficacy and potency of isoproterenol to stimulate ICa.
8. The stimulatory effect of isoproterenol on ICa was not blocked but enhanced by PKG stimulation in the presence of excess 8BrcGMP.
9. Western immunoblotting of proteins from isolated atrial and ventricular cells show significantly greater expression of PKG in atrial cells compared to ventricular cells.

The regulatory effects of intracellular cGMP on ICa are generally mediated via three pathways that include cGMP-stimulated PDE, cGMP-inhibited PDE and cGMP dependent protein kinase. In different species, and in different regions of heart, cGMP may exert different regulatory effects via its mediators and play a different role on the regulation of ICa.

Both stimulation and inhibition of basal ICa by cGMP or cGMP analogs or by NO donors have been shown by different investigators. Tohse et al. [26] have shown a small decrease of 23 and 15% by extracellular application of 300 µM and 1 mM 8BrcGMP, respectively, of basal ICa in adult rabbit ventricular cells. In contrast, Han et al. [7] showed a small increase in basal ICa by 8BrcGMP mediated via PKG in juvenile rabbit ventricular cells. Previous studies on regulation of ICa by cGMP in atrial cells are difficult to interpret. Rivet-Bastide et al. [24] have shown that basal ICa may be modulated by cGMP-stimulated PDE (PDE II) activity in human atrial myocytes. They showed that EHNA (an inhibitor of adenosine deaminase that also blocks PDE II) increased basal ICa to levels comparable to that achieved by 1 µM isoproterenol. They also showed that intracellular perfusion of cGMP also increased basal ICa by 80%. In another study, the same group of investigators [11] showed that in human atrial cells, SIN-1 (a NO donor) and cGMP increased basal ICa and suggested that cGMP-inhibited PDE (PDE III) plays a major role in regulating ICa. They also suggested the contribution of both cGMP stimulated and inhibited PDEs in the maintenance of basal cyclic nucleotide levels in human atrial myocytes. These findings on atrial cells suggested that cGMP stimulates ICa but no specific studies either inhibiting or stimulating PKG were done to assess the role of this pathway in human atrial cells. In a recent study on human atrial cells, Vandecasteele et al. [27] showed that acetylcholine decreased basal ICa by 39% and ODQ (a soluble G-cyclase inhibitor) increased ICa in 50% of the cells while SNAP (a NO donor which stimulates guanylyl cyclase) also increased basal ICa. Wang et al. [29] showed in cat atrial cells that the stimulation of ICa elicited by withdrawal of acetylcholine was blocked by methylene blue and was enhanced by preincubation with L-arginine.

The complexity of the cGMP-dependent regulation of ICa is such that both stimulation and inhibition of ICa was shown even in the same species. cGMP was shown to inhibit ICa in adult frog, rat and guinea-pig ventricular cells and in embryonic chicken ventricular cells [2,17,19,28]. The inhibitory effects of cGMP were generally attributed to either the activation of cGMP-stimulated PDE or to activation of endogenous PKG. It has also been reported that cGMP increases ICa in adult guinea-pig, frog and human myocytes [11,20,21] and the stimulatory effect was primarily attributed to the inhibition of cGMP-inhibited PDE by previous investigators. In contrast to these findings, our studies on adult and newborn rabbit ventricular cells showed that basal ICa was significantly stimulated by increased cGMP levels and inhibited by decreased cGMP levels in newborn rabbit ventricular cells [16]. This stimulatory effect of cGMP on ICa in newborn rabbit ventricular cells was shown to be mediated via PKG. However, in adult rabbit ventricular cells neither increasing nor decreasing the levels of cGMP had any significant effect on ICa.

Our results on the regulation of basal ICa by cGMP in rabbit atrial cells clearly show that increased levels of cGMP, either by the NO donor GSNO or by intracellular application of 8BrcGMP, stimulate ICa while decreased levels of cGMP, by inhibition of soluble G-cyclase (by ODQ or methylene blue) or inhibition of PKG (by KT-5823), inhibit ICa. We further show that stimulation of ICa by GSNO is not blocked in the presence of the PDE inhibitor IBMX or in the presence of the PKA inhibitor (6–22 peptide). However, stimulation of ICa by GSNO was completely blocked by PKG inhibitor KT-5823. One can argue the specificity of the compounds used to alter cGMP levels (GSNO, ODQ and MB) and for inhibiting PKG (KT-5823). To confirm the specificity of methylene blue, we showed that the inhibitory response of methylene blue was completely blocked in the presence of elevated cGMP levels on ICa. (Fig. 2). Similarly, the specificity of GSNO was confirmed by showing that the stimulatory effect of GSNO was completely blocked in the presence of ODQ and that reduced glutathione (a by-product of GSNO) had no effect on ICa. For KT-5823, we used a dose (0.6 µM) slightly higher than its Ki for PKG (0.234 µM) and much lower than its Ki for PKA (<10 µM) [10]. Therefore, we can conclude that the stimulatory effect of cGMP is mediated via PKG activation and not via PDE inhibition. Each of these modulations are similar to those seen in newborn, but not adult, ventricular cells [16].

Previous studies on the interaction of PKA and PKG dependent pathways are also difficult to interpret. Both potentiation and inhibition by cGMP or cGMP analogs or NO of β-adrenergic- or cAMP-stimulated ICa were shown by different investigators. Ono and Trautwein [21] demonstrated an additional increase by cGMP of ICa that had been elevated by isoproterenol or forskolin in guinea pig ventricular cells. In contrast, Levi et al. [17] have shown inhibition of cAMP- or 8BrcAMP-stimulated ICa in guinea pig ventricular cells by intracellular application of cGMP. The present study on the quantitative evaluation on the interaction of PKA and PKG in the regulation of ICa clearly shows that the stimulatory effect of PKA stimulation by isoproterenol was not blocked by intracellular application of 8BrcGMP and that GSNO produced additive effect under intermediate stimulation of ICa by isoproterenol. This suggests that the effects of cGMP and cAMP are not antagonistic for rabbit atrial cells. We then showed that inhibition of PKG by KT-5823 lowered basal ICa and shifted the dose response relationship of isoproterenol on ICa downward. This resulted in a much lower efficacy of isoproterenol under the condition of PKG inhibition. This suggests that PKG dependent phosphorylation of calcium channels is required in maintaining the basal ICa and is also required for the maximal stimulation of ICa by β-adrenergic stimulation.

These findings suggest that intracellular cGMP not only plays an important role in maintaining basal ICa, but may also potentiate the stimulatory effect of β-agonist stimulation of ICa. We have recently cloned and expressed [15] the cardiac form of PKG from newborn rabbit ventricle and showed that the rabbit cardiac PKG has 94% homology to previously cloned PKG sequences from either bovine lung or human lymphocytes [25,30]. Although we are not aware of any comparable studies done specifically on newborn atrial cells, it seems likely that the presence of PKG in adult atrial, but not ventricular cells represents a specific developmental decline in PKG levels in ventricular cells which does not occur in atrial cells. Such chamber specific developmental declines in ventricular, but not atrial, expression of specific proteins is known to occur for other proteins, such as myosin light chain 2a [12] and atrial natriuretic factor [1].

In summary, these findings suggest that in adult rabbit atrial cells, intracellular cGMP exerts a stimulatory effect on basal ICa and that this regulation is most likely mediated via a PKG-dependent phosphorylation pathway and is independent of PKA and PDEs. The adult rabbit atrial cells may have a phosphorylation site associated with the calcium channel which can be phosphorylated by PKG and this site, when phosphorylated, increases the availability of L-type calcium channels and is involved in maintaining basal ICa. Since the effects of cGMP are not antagonistic to cAMP, it is possible that PKG and PKA phosphorylation sites of calcium channels are similar in rabbit atrial cells. PKG is highly expressed in atrial cells and PKG dependent phosphorylation may be necessary for the maintenance of basal ICa and for maximal stimulation of ICa by β-adrenergic stimulation in atrial cells.

Time for primary review 32 days.


   Acknowledgments
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
References
 
This work was partially supported by NIH grant HL49438 (Dr. Joyner), grant-in-aid from American Heart Association (Dr. Kumar), NIH grant HL56787 (Dr. Kumar), The Children's Heart Center and by the Emory Egleston Children's Research Center. The antibody for PKG was generously supplied by Dr. Thomas Lincoln, Department of Pathology, University of Alabama at Birmingham.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 

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