© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
Protein kinase G reverses all isoproterenol induced changes of cardiac single L-type calcium channel gating
Department of Cardiology and Angiology, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany
* Corresponding author. Tel.: +49-511-532-3841; fax: +49-511-532-5412 schroeder.f{at}mh-hannover.de
Received 21 February 2000; accepted 11 July 2000
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
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Objective: cGMP reduces the effect of β-adrenoceptor agonists on cardiac L-type calcium current by protein kinase G activation. Stimulation of β-adrenoceptors increases protein kinase A dependent phosphorylation of L-type calcium channels via cAMP. At the single channel level, protein kinase A dependent phosphorylation increases both availability and open probability. The present study investigates how cGMP antagonises protein kinase A induced changes of single L-type calcium channel gating. Methods: Single L-type calcium channels were recorded in the cell attached configuration of the patch clamp technique in isolated mouse ventricular myocytes. Results: The β-adrenoceptor agonist isoproterenol (10–6 M) enhanced single channel peak average current by increasing availability and open probability and decreasing the time constant of long close times. 8-Br-cGMP (10–3 M) completely reversed these effects. The phosphatase inhibitor okadaic acid (10–6 M) did not influence the effect of 8-Br-cGMP. The protein kinase G inhibitor Rp-8Br-PET-cGMPS (10–7 M) abated the effect of 8-Br-cGMP. Activation of protein kinase A by the hydrolysis-resistant cAMP derivative 8-Br-cAMP (10–3 M) enhanced L-type calcium channel activity like isoproterenol and its effect was also reversed by 8-Br-cGMP. Conclusion: 8-Br cGMP diminishes β-adrenoceptor activation of L-type calcium channels via protein kinase G. It interacts with the β-adrenoceptor signaling pathway distal of adenylyl cyclase. Our observations suggest that protein kinase G interacts either with protein kinase A or directly with the L-type calcium channel.
KEYWORDS Signal transduction; Protein kinases; Adrenergic (ant)agonists; Ca-channel; Myocytes; Single channel currents
This article is referred to in the Editorial by F. Chen (pages 362–364) in this issue.
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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In cardiomyocytes cGMP is the intracellular second messenger of various extracellular stimuli such as atrial natriuretic peptide and nitric oxide [1–3]. In heart failure both are increased [4,5] due to a rise in cytokines, which in turn stimulate the expression of inducible nitric oxide synthase in the failing heart [6]. Thus, NO and its second messenger cGMP appear to contribute to the lack of effect of β-adrenergic stimulation in heart failure [6]. Accordingly, the L-type calcium channel as an important target of cGMP is of major interest. Numerous studies have addressed this issue in various animal models, mostly by measuring whole-cell calcium current of isolated cardiomyocytes. However, the findings are inconsistent. Some authors describe a decrease of basal L-type calcium current after application of stabilised cGMP-derivates [7,8]. Others outline that cGMP only decreases L-type calcium current after the cell has been stimulated by β-adrenoceptor agonists [9].
Hitherto, no work focused on a detailed single channel analysis of cardiac L-type calcium channels as influenced by cGMP. Analysis of single channel gating provides a more detailed insight into channel regulation, because the whole cell current I is a function of both, the number of functional channels n and their individual properties i (single channel current amplitude), open probability (Popen, fraction of time spent in the open state during active sweeps), and availability (factive, fraction of active sweeps per number of test pulses), where
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The latter two parameters are known to be differentially regulated by β-adrenergic induced channel phosphorylation and phosphatase induced channel dephosphorylation [10–12]. These differential effects on the very same molecular target can be explained by distinct pathways of signal transduction and different regulatory sites on the channel.
Hence, we addressed the following questions: First, how does cGMP influence single L-type calcium channel gating qualitatively? Second, is it possible to localise the molecular target of intracellular cGMP leading to a decrease of L-type calcium current?
Since there is increasing interest in genetically engineered mice, effort has been made to characterise whole cell membrane currents of murine wild type cardiomyocytes [13]. However, a detailed single L-type calcium channel analysis has not been performed yet.
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2 Methods |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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2.1 Isolation of ventricular myocytes
Our 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). Ventricular myocytes from adult male mice (16–24 g) were isolated by enzymatic dissociation using the method described elsewhere [14]. In brief, mice were heparinised (5000 units/kg body weight) by intraperitoneal injection 20 min before being killed by cervical dislocation. The heart was quickly excised, mounted on a Langendorff apparatus and perfused with a nominally Ca2+-free, oxygenated preparation buffer (in mM: NaCl 133.5, KCl 4.0 NaH2PO41.2, MgSO41.2, HEPES 10.0, bovine serum albumin 1 mg/ml; pH 7.4, 37°C). After 5 min the perfusion solution was switched to preparation buffer containing collagenase (Worthington type I; 67 U/ml) and 25 µM CaCl2. After 9–13 min of tissue digestion the hearts were removed from the Langendorff apparatus and ventricular tissue was cut into small chunks that were gently agitated in preparation buffer containing 100 µM Ca2+. In order to remove the collagenase and to increase extracellular Ca2+ gradually, myocytes were washed twice. During this procedure the cells were permitted to settle under gravity. The supernatant was removed and the cells were resuspended in preparation buffer containing 200 µM and 500 µM Ca2+, respectively. Cells were stored at room temperature until use within 10 h. Before electrophysiological measurements aliquots of cell suspension were preincubated at room temperature for 30–60 min with 10 µM BAPTA-AM (1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl)-ester, Calbiochem). This procedure yielded 10–20% of elongated and striated myocytes.
2.2 Measurements of barium currents through single channels
Cells were placed in disposable perfusion chambers containing 1–2 ml of bath solution (in mM: K-glutamate 120, KCl 25, MgCl2 2, HEPES 10, EGTA 2, CaCl2 1, Na-ATP 1, dextrose 10, pH 7.4, with NaOH, room temperature). Single channel recordings were performed in the cell-attached configuration [15]. In brief, pipettes (borosilicate glass, 7–10 M
) contained (mM) BaCl2 70, sucrose 110, HEPES 10 (pH 7.4 with TEA-OH). Barium was used as charge carrier to avoid calcium induced calcium current inactivation. Barium currents were elicited for 150 ms at 1.66 Hz by depolarising test pulses with command pulses from –100 to +20 mV. Recordings were achieved at 10 kHz and filtered at 2 kHz (–3 dB, 4-pole Bessel) using an Axopatch 200 B amplifier (Axon Instruments, Foster City, CA, USA). The PClamp software (version 6.0, Axon Instruments) was used for data acquisition and analysis of openings and closures. L-Type calcium channels (identified by their typical unitary conductance and voltage dependence of activation) were usually found in one out of ten to fifteen patches.
2.3 Drug solutions
Isoproterenol (from Sigma, 10–4 M stock in ascorbic acid), 8-Br-cGMP (from Sigma, 10–1 M stock), 8-Br-cAMP (from Sigma, 10–1 M stock in DMSO), okadaic acid (NH4 salt from Calbiochem, 10–4 M stock in DMSO), Rp-8-Br-PET-cGMPS (from Biolog, 10–5 M stock in 1% DMSO), Bay K 8644 (from Sigma, 10–4 M stock in 10% DMSO) were added to the bath as a 10–20 µl bolus. Final concentrations reached target concentrations ±15% deviation depending on the exact amount of the bath volume, which was determined after each experiment. In experiments with two or more test compounds, substances were applied in succession (except isoproterenol and okadaic acid, which were applied simultaneously).
2.4 Data analysis and statistics
Linear leak and capacity currents were digitally subtracted using averaged currents of non-active sweeps. The availability (fraction of sweeps containing at least one channel opening, e.g. fraction of active sweeps per total number of test pulses), P0 (i.e. fractional occupancy of the open state during active sweeps), and the peak ensemble average current were corrected by the number of channels in the patch (n). Peak current is the maximum of the ensemble average current from one single channel. In case of double- or triple-channel patches, n was derived from the maximum current amplitude observed divided by the unitary current amplitude. Peak current was normalised by division through n. The availability was corrected by the square root method: (1–availabilitycorr) is the nth root of (1–availabilityuncorr). Availabilitycorr and availabilityuncorr are corrected and uncorrected availability, respectively. The corrected P0 was calculated on the basis of the corrected number of active sweeps, i.e. total open time (in ms) within all sweeps of the ensemble, divided by (150 msxnxavailabilitycorrxnumber of test pulses). Openings and closures were identified by the half-height criterion. Mean open times were calculated from the total open time of the channel divided by the sum of the number of closures and the number of active sweeps. Mean close time was calculated from total close time divided by the number of closures. Close time distributions were analysed after binning (bin width 1 ms) by using the maximum likelihood method (PStat version 6.0). Open and close times, first latency, and burst length were analysed only when the patches contained one single channel. Burst length is defined as the interval between the first opening and the last closure of an active sweep.
Data are presented as the mean and SEM of n observations. Significance was checked by 1-way ANOVA analysis and Bonferroni posttest at the level of P<0.05.
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3 Results |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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3.1 Single channel conductance
Single L-type calcium channels from mouse cardiomyocytes were characterised by their conductance. Depolarising pulses from a holding potential of –100 mV were elicited to test potentials between 0 and +30 mV. Channel availability and open probability increased with voltage. We determined the single channel conductance by plotting the amplitude of apparently fully resolved openings against the test potential for every single experiment. The mean of the slopes of the linear relationships of four experiments is 17.3±3.1 pS. In order to facilitate the occurrence of fully resolved openings at all investigated test potentials, we induced long channel openings by applying 10–6 M of the calcium channel agonist Bay K 8644 (Fig. 1). In three experiments long openings were analysed at different test potentials. The mean slope of the linear I/V relationship was 20.5±2.1 pS. These results are comparable with data previously obtained in human ventricular myocytes under the same conditions [16]. We consider that the channels are L-type calcium channels because of this typical single channel conductance and the propensity of Bay K 8644 to induce long channel openings.
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3.2 Effect of 8-Br-cGMP on single channel gating
To examine the effect of the hydrolysis-resistant 8-Br-cGMP on basal L-type channel activity, single channel recordings were performed under control conditions and after addition of 8-Br-cGMP (10–3 M). Despite the rather high concentration of 8-Br-cGMP we did not see any effect on basal single channel gating (Table 1, Fig. 2).
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However, after β-adrenergic stimulation of the cardiomyocytes by isoproterenol (10–6 M) 8-Br-cGMP (10–3 M) reversed all isoproterenol induced changes in channel gating (Fig. 3, Table 2). Isoproterenol increased ensemble average current by a rise in channel availability and open probability. The increase in open probability was due to a decrease in the time constant of long close times (
close,long). The opposite effect concerning these parameters was observed when 8-Br-cGMP was given after treatment with isoproterenol (Fig. 4). The decrease in first latency and the increase in burst length as typical isoproterenol induced alterations in L-type calcium channel gating were not significantly changed by 8-Br-cGMP. However, there was a strong trend to reverse these isoproterenol induced changes of channel gating.
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3.3 Effect of 8-Br-cGMP is mediated by protein kinase G
To test whether the effect of 8-Br-cGMP on the single L-type calcium channel was mediated by protein kinase G, we applied the protein kinase G inhibitor (Rp)-8-Br-PET-cyclic GMPs (10–7 M) after stimulation of single channel activity with isoproterenol. The concentration of (Rp)-8-Br-PET-cyclic GMPs we used has already been shown to be effective in inhibiting protein kinase G in bovine lung (Ki (PKG)=3.5x10–8 M, Ki (PKA)=1.1x10–5 M [17]). Both, the fact that the concentration we used was 100 fold lower than Ki (PKA) and the fact that (Rp)-8-Br-PET-cyclic GMPs did not alter the effect of isoproterenol precludes simultaneous inhibition of protein kinase A. (Rp)-8-Br-PET-cyclic GMPs entirely abated the antagonistic feature of 8-Br-cGMP to isoproterenol stimulation (Fig. 5), indicating that 8-Br-cGMP exerts its effect via stimulation of protein kinase G.
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3.4 Target of protein kinase G
Channel availability is known to be controlled by a type 1 protein phosphatase [12] and this phosphatase is itself controlled by cAMP [18], whereas open probability seems to be regulated by a type 2a phosphatase [12]. To examine whether cGMP antagonises the effect of β-adrenergic stimulation of single L-type calcium channels by activation of phosphatases we applied isoproterenol and okadaic acid (10–6 M) in a concentration known to inhibit activity of both type I phosphatase and type 2a phosphatase [19]. However, okadaic acid did not influence the effect of 8-Br-cGMP (Fig. 6). Therefore, an increase of channel dephosphorylation by phosphatases seems to be unlikely.
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Fig. 7 shows the effect of 8-Br-cGMP after activation of protein kinase A with the hydrolysis-resistant cAMP derivative 8-Br-cAMP (10–3 M). 8-Br-c-AMP enhanced L-type calcium channel activity like isoproterenol, i.e. increase of open probability and availability. Likewise, 8-Br-cGMP reversed these changes of channel gating.
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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In cardiomyocytes cGMP plays a role antagonistic to that of cAMP. Cyclic-AMP as the second messenger of the β-adrenergic signaling pathway activates protein kinase A, thereby phosphorylating the L-type calcium channel. Thus, ensemble average current increases due to a rise in channel open probability and availability. For cGMP the exact mechanism of reducing β-adrenergic stimulated ICa,L has not yet been studied on single channel level. Our data show that the opposing roles of cAMP and cGMP in heart are not only limited to force of contraction [20] and whole cell calcium current [21], but can also be applied to single L-type calcium channel level:
- 1. 8-Br-cGMP does not influence gating properties of basic cardiac L-type calcium channel in mice ventricular myocytes.
- 2. However, after β-adrenergic stimulation by isoproterenol 8-Br-cGMP reverses the typical isoproterenol induced changes in ensemble average current, open probability, availability, first latency, burst length and time constant of long close times.
- 3. This effect was mediated by activation of protein kinase G and not due to either PKG-dependent activation of phosphatases 1 and 2a or activation of phosphodiesterases.
- 2. However, after β-adrenergic stimulation by isoproterenol 8-Br-cGMP reverses the typical isoproterenol induced changes in ensemble average current, open probability, availability, first latency, burst length and time constant of long close times.
Our data confirm results of whole cell voltage clamp experiments revealing that protein kinase G is the target protein of cGMP in mammalian heart [7,9,22]. The results concerning the effect of cGMP on basal ICa,L are controversial. Sumii and Sperelakis show a decrease in rat basal ICa,L [7] and a decrease in single channel activity of chicken and rabbit ventricular myocytes [23,24], whereas others could not see any effect of cGMP on basal ICa,L in adult rat [9] and frog ventricular myocytes [21]. This could be due to different basal levels of cAMP or protein kinase A activity on account of species and age differences of the used animal models. Our data do not show any effect of 1 mM 8-Br-cGMP on single L-type calcium channels. However, after β-adrenergic stimulation all typical isoproterenol induced changes in L-type calcium channel gating, e.g. ensemble average current, open probability, availability, close, long [25,26] are reversed to control levels by 8-Br-cGMP. Other parameters, e.g. burst length and first latency, are not significantly changed, but show a strong trend. This functional antagonism could be explained by interference of protein kinase G with the the β-adrenergic signaling pathway. Since 8-Br-cGMP reduces the effect of hydrolysis-resistant 8-Br-cAMP to the same extent as isoproterenol, the target protein of protein kinase G is localised distal to adenylate cyclase and phosphodiesterases in the β-adrenergic signaling cascade. Protein kinase G dependent phosphorylation and thereby inhibition of protein kinase A could explain the lack of effect of 8-Br-cGMP on basal Ica as well as the reverse influence of isoproterenol and 8-Br-cGMP on single channel gating properties. Accordingly 8-Br-cGMP is suggested to reduce the phosphorylation state of L-type calcium channels by inhibiting protein kinase A via protein kinase G activation. Our functional analysis is consistent with biochemical data of Geahlen et al. revealing protein kinase A as a substrate of protein kinase G [27].
Both, the lack of effect of 8-Br-cGMP on basal L-type calcium channels and the complete reversal of isoproterenol induced channel gating properties by 8-Br-cGMP, could alternatively be explained by direct phosphorylation of the L-type calcium channel by protein kinase G following channel phosphorylation of protein kinase A [28]. However if so, a completely opposing action of protein kinase A and protein kinase G on L-type calcium channel gating would be unlikely.
In summary, in mouse ventricular myocytes 8-Br-cGMP reverses all isoproterenol or 8-Br-cAMP induced changes in single L-type calcium channel gating. This effect is mediated by protein kinase G and not due to phosphatase activation. The opposing action of both second messengers on all gating properties suggests a protein kinase G dependent protein kinase A inhibition of L-type calcium channel. The present paper shows a short-term effect of protein kinase G activation on the regulation of L-type calcium channels. Since iNOS induction may permanently activate the NO/cGMP-signaling pathway in human heart failure, further studies using genetically engineered mouse models, e.g. protein kinase G overexpressing mice, are needed to assess the long-term effect of protein kinase G activation in regulating cardiac L-type calcium channels.
Time for primary review 18 days.
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