Skip Navigation

Cardiovascular Research 1997 34(2):360-367; doi:10.1016/S0008-6363(97)00043-6
© 1997 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Martynyuk, A.E
Right arrow Articles by Rankin, A.C
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Martynyuk, A.E
Right arrow Articles by Rankin, A.C
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 1997, European Society of Cardiology

Role of nitric oxide, cyclic GMP and superoxide in inhibition by adenosine of calcium current in rabbit atrioventricular nodal cells

A.E Martynyuka, K.A Kaneb, S.M Cobbea and A.C Rankina,*

aDepartment of Medical Cardiology, Royal Infirmary, Glasgow, G31 2ER, UK
bDepartment of Physiology and Pharmacology, University of Strathclyde, Glasgow, G1 1XW, UK

* Corresponding author. Tel.: +44 (141) 211 4833; fax: +44 (141) 552 4683.

Received 22 July 1996; accepted 30 December 1996


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: To study the intracellular pathways which mediate the inhibitory actions of adenosine on isoprenaline-stimulated calcium current (ICa) in atrioventricular (AV) nodal myocytes. Methods: The whole-cell patch-clamp technique was used to record ICa from rabbit AV nodal cells, isolated by enzymatic and mechanical dispersion. Results: Isoprenaline, 0.1 µM, increased peak ICa from 0.58±0.08 to 1.23±0.1 nA, and this increase was reversibly inhibited by adenosine, 10 µM (83±6%), which we have previously shown to be mediated by nitric oxide (NO) production. A membrane-permeable analogue of cyclic GMP, 8-Br-cGMP (300 µM), an inhibitor of cGMP-stimulated phosphodiesterase, prevented the effect of adenosine on ICa. Methylene blue (10 µM), an inhibitor of NO-sensitive guanylyl cyclase and a generator of superoxide (·O2–), did not prevent, but increased, the inhibiting action of adenosine (49.5±6.6%, P<0.01). Methylene blue (50 µM) caused a reduction of ICa, with further inhibition when combined with adenosine. A ·O2–-generating system, xanthine oxidase (0.02 U/ml) and purine (2.3 mM), also increased the inhibitory action of adenosine on ICa. Inhibition of ICa by adenosine in the presence of xanthine oxidase was not prevented by 8-Br-cGMP (300 µM) and was not influenced by pre-incubation of cells with a NO synthase inhibitor, L-NAME (0.5 mM). Conclusions: The inhibitory effect of adenosine on ICa in rabbit AV nodal myocytes can be mediated by two mechanisms—stimulation of cGMP-stimulated phosphodiesterase by NO-induced cGMP, and a mechanism which involves interaction with ·O2– production.

KEYWORDS Adenosine; Rabbit, heart; Atrioventricular nodal cells; Nitric oxide; cGMP; Superoxide; Calcium current


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Adenosine has a variety of regulatory roles on cardiac functions, including negative chronotropic [1], inotropic [2]and dromotropic actions [3]. The negative dromotropic action, with block of atrioventricular (AV) nodal conduction, is of clinical importance because of the pharmacological use of adenosine in the treatment of cardiac arrhythmias [4]and the pathophysiological role of endogenous adenosine in the development of heart block in patients with acute myocardial infarction [5]. We have previously studied the ionic mechanisms underlying this action in isolated rabbit AV nodal myocytes [6, 7]. Adenosine directly activated an inwardly rectifying potassium current (IK(Ado)), and also had an indirect inhibitory action on the L-type calcium current (ICa) following stimulation by catecholamines. The intracellular transduction mechanisms underlying this latter anti-adrenergic action of adenosine are complex and we have shown that they include A1-receptor-mediated generation of nitric oxide (NO) [7]. The aim of the present work was to study the subsequent mechanisms by which NO production resulted in inhibition of ICa.

Many cardiovascular effects of NO are mediated through stimulation of guanylate cyclase and production of guanosine 3',5'-cyclic monophosphate (cGMP). Involvement of cGMP in the modulation of calcium current by NO, following application of NO donors, has been demonstrated in frog and guinea-pig ventricular myocytes [8, 9]and human atrial myocytes [10]. However, the subsequent mechanisms differ in different tissues and there is evidence that the modulation by NO of ICa can be due to either activation of the cGMP-stimulated phosphodiesterase (cGS-PDE) [8, 11]or activation of protein kinase G (PKG) [9]or via inhibition of the cGMP-inhibited phosphodiesterase (cGI-PDE) [10].

The intracellular pathways which mediated the inhibitory actions of adenosine on isoprenaline-stimulated calcium current (ICa) were studied using compounds which are known to inhibit components of the transduction mechanisms. This approach is complicated by the fact that such compounds may have multiple actions. The stable analogue of cGMP, 8-Br-cGMP, can be used as an activator of PKG [12, 13]but it can also inhibit cGS-PDE [14]. Methylene blue is an inhibitor of NO-sensitive guanylyl cyclase, but this effect is dependent on the generation of superoxide [15, 16], which may have additional actions independent of cGMP. The aim of this work, therefore, was to determine the role of NO and cGMP in the actions of adenosine and to assess whether other mechanisms, in particular those dependent on superoxide production, might also play a role.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Preparation of isolated cells from the rabbit atrioventricular node
Single isolated AV nodal myocytes were prepared from rabbit hearts using a method which combines enzymatic and mechanical dispersion of the tissue, as described previously [6, 7]. In brief, the excised hearts from New Zealand white male rabbits (2.5–3.5 kg) were retrogradely perfused with enzyme solution containing 0.8 mg ml–1 collagenase (Type 1, Worthington) and 0.08 mg ml–1 protease (Type XIV, Sigma) for 5–10 min. The region of the AV node was then removed, and cells were isolated by gentle agitation. The isolated myocytes were stored at 4°C in high-K low-Ca solution until use.

The investigation was performed in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986, published by Her Majesty's Stationery Office, London.

2.2 Solutions and chemicals
The basic solution for cell isolation contained (in mM): 130 NaCl, 4.5 KCl, 3.5 MgCl2, 0.4 NaH2PO4, 5 HEPES, 10 glucose, 20 taurine, 10 creatine, pH 7.25. The composition of the high-K, low-Ca storage solution was (in mM): 100 L-glutamic acid, 30 KCl, 10 HEPES, 0.5 EGTA, 5 taurine, 20 glucose, 5 MgCl2, 5 succinic acid, 5 creatine, 2 Na2ATP, 5 β-OH-butyric acid, pH 7.2. The HEPES-buffered Tyrode's solution for superfusion of cells in the recording chamber was made fresh daily and contained (mM): 130 NaCl, 2.7 KCl, 2 CaCl2, 1 MgCl2, 10 Glucose, 5 HEPES, pH adjusted to 7.4 with NaOH at 35–37°C. Depending upon experiments, solutions were modified by appropriate addition of compounds and drugs. All compounds were diluted into the normal Tyrode's solution immediately prior to use. All chemicals and drugs used in the experiments were purchased from Sigma Chemical Co. (Dorset, UK).

The pipette-filling solution was potassium-free, replaced by caesium, to block outward potassium currents and had the following composition (mM): 130 CsCl, 1 MgCl2, 5 HEPES, 5 EGTA, 4 Na2-ATP, 0.4 Na-GTP; pH was adjusted to 7.25 with CsOH.

2.3 Electrophysiological techniques
The whole-cell configuration of the patch-clamp technique was used to record high-threshold calcium current. The patch microelectrodes were pulled from 1.5 mm borosilicate glass (Clark Electromedical) using a Narishige two-stage vertical puller. When filled with recording solution, patch microelectrodes had resistance of 2–5 M{Omega}. Aliquots of the cell suspension were transferred to the recording chamber, which was mounted on the stage of an inverted Nikon microscope. The isolated cells were allowed to settle for 15 min before superfusion with normal Tyrode solution was started. The temperature of the bath was maintained at 35–37°C using a modified Temperature Controlled Perfusion System (Life Science Resources Limited, Cambridge, UK). Solutions were superfused at approximately 1.5 ml/min with a gravity feed system, giving an exchange time of approximately 30 s.

Spontaneously beating, spindle-shaped myocytes isolated from the region of the AV node were studied. We have previously described the electrophysiology of these cells, which exhibited spontaneous action potentials and were smaller, relatively depolarised and had higher membrane resistance compared to atrial cells [6]. The use of caesium-containing pipettes prevented confirmation of these properties in the present study. Whole-cell membrane capacitance was determined from the charging curves in response to 10 mV steps as 44±1.5 pF (n=23). Whole-cell voltage-clamp experiments were performed using an Axopatch 1D (Axon Instruments, Inc., USA). The high-threshold calcium current was elicited by 50–100 ms square pulse from a holding potential of –50 to 10 mV at 0.1 Hz. Peak inward current was measured relative to the holding current. Effects of drugs on the peak inward current were expressed as percentage of the inward current prior to that drug application. The records were monitored on an oscilloscope (Model DTS 20) and were digitised on-line using a National Instruments LAB-PC analogue-to-digital board and stored on the hard disc of an IBM-compatible PC (Viglen IV/25). Voltage clamp experimental protocols and off-line data analysis were performed using the software program ‘WCP’, written by John Dempster (Department of Physiology and Pharmacology, University of Strathclyde, Glasgow).

2.4 Statistics
Values are presented as mean±s.e.m. Statistical significance was evaluated by Student's unpaired t-test, and P<0.05 was considered significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effect of adenosine on isoprenaline-stimulated calcium current
The increase in the magnitude of peak ICa produced by isoprenaline (0.1 µM), and the reversible inhibitory action of the subsequent addition of adenosine (10 µM) is shown in Fig. 1. Isoprenaline (0.1 µM) induced a mean increase in peak ICa from 0.58±0.08 nA to 1.23±0.1 nA (n=36). The mean ICa when 10 µM adenosine was added to 0.1 µM isoprenaline was reduced to 83.7±6% of the amplitude before application of adenosine (n=7).


Figure 1
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Inhibition by adenosine of isoprenaline-induced increase in ICa in a rabbit atrioventricular (AV) nodal cell. The time course of changes in peak ICa in response to isoprenaline (0.1 µM) and adenosine (10 µM) is shown, with horizontal bars denoting the time during which each drug was applied. The individual current records (A–D) correspond to the marked time points.

 
3.2 Effect of 8-Br-cGMP on the anti-adrenergic effect of adenosine
The membrane-permeable stable analogue of cGMP, 8-Br-cGMP, inhibits cGS-PDE [14]and activates PKG with high relative affinities [12, 13]. Fig. 2 shows that 8-Br-cGMP, 300 µM, when added after isoprenaline (0.1 µM), had no effect on peak ICa. The mean peak ICa was 102.8±2.8% (n=4) of the amplitude before application of 8-Br-cGMP, indicating that PKG activation did not attenuate isoprenaline-stimulated ICa in rabbit AV nodal cells. However, 8-Br-cGMP (300 µM) prevented inhibition by adenosine of the isoprenaline-stimulated ICa (Fig. 2). The mean peak ICa was 99.8±2.6% (n=4) of the amplitude before application of 8-Br-cGMP and adenosine. These findings suggest that the inhibitory actions of adenosine on isoprenaline-stimulated ICa are mediated by cGMP and its activation of a cGS-PDE.


Figure 2
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Prevention by 8-Br-cGMP of the inhibition by adenosine of the isoprenaline-stimulated ICa in a rabbit AV nodal cell. Changes in peak ICa in response to isoprenaline (0.1 µM), 8-Br-cGMP (300 µM) and adenosine (10 µM) are shown. The horizontal bars denote the time during which each drug was applied. The individual current records (A–D) correspond to the marked time points.

 
3.3 Effects of methylene blue and adenosine on ICa
Methylene blue has been used as a blocker of the NO-sensitive guanylyl cyclase [8, 11, 17], and therefore would be expected to block the inhibitory anti-adrenergic action of adenosine. Fig. 3 shows that this was not the case, but rather that the inhibitory action of adenosine (10 µM) was augmented and irreversible. Methylene blue (10 µM) itself had no significant effect on isoprenaline-augmented ICa (107±3.3% of prior peak ICa). The subsequent application of 10 µM adenosine reduced the peak ICa to 49.5±6.6% of its amplitude before the application of methylene blue and adenosine (n=8, P<0.01). The inhibitory effect of adenosine in combination with methylene blue was partially reversible in 2 cells and irreversible in the others. These results indicated a different mechanism, independent of cGMP, underlying the inhibitory action of adenosine in the presence of methylene blue.


Figure 3
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Potentiation of the inhibitory effect of adenosine on ICa in the presence of methylene blue. Changes in peak ICa in response to application of isoprenaline (0.1 µM), methylene blue (10 µM) and adenosine (10 µM) are shown. The horizontal bars denote the time during which each drug was applied. The individual current records (A–D) correspond to the marked time points.

 
Fig. 4 shows that a higher concentration (50 µM) of methylene blue alone also had an inhibitory action on ICa. There was further irreversible inhibition of ICa when methylene blue (50 µM) was combined with adenosine (10 µM). The mean peak ICa with 50 µM methylene blue was 80±7.7% of the amplitude before its application (n=6) and was further reduced to 52±8% by the addition of 10 µM adenosine. A possible explanation for these inhibitory actions of methylene blue and its combination with adenosine may be related to the additional ability of methylene blue to generate the superoxide radical, ·O2– [12, 20].


Figure 4
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Inhibition of isoprenaline-stimulated ICa by methylene blue. A higher concentration of methylene blue (50 µM) inhibited the peak ICa which had been increased by isoprenaline (0.1 µM). Following reduction of ICa by methylene blue (50 µM) there is a further marked irreversible reduction of ICa by exposure to the combination of methylene blue (50 µM) and adenosine (10 µM). The horizontal bars denote the time during which each drug was applied. The individual current records correspond to the marked time points.

 
3.4 Effects of superoxide generation on ICa and action of adenosine
To determine whether superoxide (·O2–) could indeed be involved, the cells were exposed to xanthine oxidase which, in the presence of a suitable substrate such as purine (7H-imidazo[4,5-d]pyrimidine), generates ·O2– [18, 19]. The addition of xanthine oxidase (0.06 U/ml) and purine (2.3 mM) reduced the isoprenaline-stimulated ICa (Fig. 5) to 41±8.6% (n=4). The effect was dependent on the concentration of xanthine oxidase, in combination with the same purine concentration (2.3 mM), with less inhibition of ICa produced by 0.02 U/ml of xanthine oxidase (88.2±4.7%, n=5). However, similar to a low concentration of methylene blue, there was a marked inhibitory effect of adenosine in the presence of 0.02 U/ml of xanthine oxidase (Fig. 6). The mean peak ICa was reduced to 47.2±8% of the amplitude before the application of xanthine oxidase and adenosine (n=3, P<0.01).


Figure 5
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Inhibition of isoprenaline-induced ICa by superoxide. The increase in ICa in response to isoprenaline (0.1 µM) was inhibited by the combination of xanthine oxidase (0.06 U/ml) and purine (2.3 mM), which generates superoxide. Changes in peak ICa are shown. The horizontal bars denote the time during which each drug was applied. The individual current records correspond to the marked time points.

 

Figure 6
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Increased inhibitory effect of adenosine on ICa in the presence of lower concentration of xanthine oxidase. The increase in peak ICa in response to isoprenaline (0.1 µM) was not changed by a lower concentration of xanthine oxidase (0.02 U/ml) with purine (2.3 mM) but was markedly inhibited by the addition of adenosine (10 µM). The horizontal bars denote the time during which each drug was applied. The individual current records (A–D) correspond to the marked time points.

 
Evidence was sought to support the hypothesis that this was an additional action of adenosine which was independent of NO and cGMP production. Fig. 7 shows that 8-Br-cGMP, 300 µM, did not prevent the inhibitory effect of adenosine on ICa in the presence of xanthine oxidase and purine (mean ICa reduced to 46.1±3.8%, n=4). We have previously shown that pre-incubation of the AV nodal myocytes with the NO synthase inhibitor, L-NAME (0.5 mM), prevented the attenuation by adenosine of the isoprenaline-stimulated calcium current [7]. Fig. 8 shows that L-NAME did not prevent the inhibition of ICa by adenosine in the presence of a low concentration of xanthine oxidase (0.02 U/ml) with purine, which was confirmed in 3 cells. These results suggest that the inhibitory action of adenosine in the presence of superoxide generation involves a mechanism which is independent of NO and cGMP production.


Figure 7
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 The lack of effect of 8-Br-cGMP on the inhibiting action of adenosine on ICa in the presence of xanthine oxidase and purine. The increase in peak ICa in response to isoprenaline (0.1 µM) was unaffected by xanthine oxidase (0.02 U/ml) and purine (2.3 mM) and was inhibited by adenosine (10 µM) in the presence of 8-Br-cGMP (300 µM). The horizontal bars denote the time during which each drug was applied. The individual current records (A–D) correspond to the marked time points.

 

Figure 8
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8 Lack of effect of pre-incubation of myocytes with L-NAME, a NO synthase inhibitor, on the actions of xanthine oxidase, purine and adenosine on isoprenaline-increased ICa. Pre-incubation with L-NAME did not prevent the inhibition of isoprenaline-increased ICa by adenosine (10 µM) in the presence of xanthine oxidase (0.02 U/ml) and purine (2.3 mM). The horizontal bars denote the time during which each drug was applied.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In this study we have demonstrated, in line with our previous results [7], that adenosine attenuated the isoprenaline-induced increase in ICa in rabbit AV nodal cells. This anti-adrenergic effect of adenosine was shown previously to be blocked by the NO synthase inhibitor, L-NAME, suggesting that it was mediated via the production of NO [7]. The findings of the present study indicate that this effect of adenosine on isoprenaline-stimulated calcium current involves the activation of guanylyl cyclase and the production of cGMP.

The mechanisms underlying the modulation by cGMP of cardiac calcium current have been shown to include activation of either cGMP-dependent protein kinase G (PKG) [9]or cGMP-stimulated phosphodiesterase (cGS-PDE) [8, 11], or inhibition of the cGMP-inhibited phosphodiesterase (cGI-PDE) [10], in different tissues. In the present findings with rabbit AV nodal myocytes, 8-Br-cGMP, a membrane-permeable and stable analogue of cGMP which activates protein kinase G (PKG) [12, 13], did not modify the isoprenaline-stimulated ICa. This suggested that activation of this enzyme was not a necessary step in the attenuation of ICa. However, 8-Br-cGMP did prevent the adenosine attenuation of the isoprenaline-stimulated ICa. An explanation for this finding is that 8-Br-cGMP can also inhibit the cGMP-stimulated phosphodiesterase (cGS-PDE), as recently reported [14]. If adenosine activates guanylyl cyclase, following the production of NO, the subsequent stimulation of cGS-PDE would enhance the breakdown of isoprenaline-elevated cAMP leading to a reduction of the isoprenaline-stimulated ICa. In the presence of 8-Br-cGMP, cGS-PDE may be inhibited, thus preventing the anti-adrenergic effect of adenosine. Support for these conclusions comes from recent evidence that a similar mechanism involving cGS-PDE may underlie the cholinergic modulation of calcium current in rabbit AV nodal cells [18].

A similar signal transduction mechanism has been proposed to explain the anti-adrenergic effect of muscarinic stimulation in frog ventricular myocytes [8]and rabbit sino-atrial (SA) nodal cells [11]. It is of interest to note, however, that 8-Br-cGMP did not block the anti-adrenergic effect of carbamylcholine in SA nodal cells [11]. This could be explained by the fact that 8-Br-cGMP is a competitive inhibitor of cGS-PDE [14]and its blocking activity would, in part, be dependent upon the level of cGMP. In our experiments, adenosine attenuated but did not completely inhibit the isoprenaline stimulated ICa, whereas in the experiments of Han et al. [11]the inhibition by carbamylcholine was total. It is possible, therefore, that in the two sets of experiments, a different level of stimulation of guanylyl cyclase was achieved resulting, in our experiments, in a lower level of cGMP which was more readily blocked by 8-Br-cGMP.

Another important finding of this study was that under certain conditions the reduction in ICa by adenosine in AV nodal cells may be unrelated to activation of NO synthase but was related to interaction with superoxide (·O2–) production. In the presence of concentrations of xanthine oxidase and purine, or methylene blue, which by themselves did not reduce ICa, the anti-adrenergic effect of adenosine was potentiated. However, neither 8-Br-cGMP nor L-NAME prevented the inhibition in ICa produced by a combination of adenosine and xanthine oxidase plus purine. Xanthine oxidase in the presence of a suitable substrate, in this case purine, reduces O2 and produces the superoxide radical, ·O2– [19–21]. Similarly, methylene blue can generate superoxide and other reactive oxygen species [15, 16]. In higher concentrations both xanthine oxidase and methylene blue by themselves inhibited the isoprenaline-stimulated current. This may reflect the ability of free radicals to modify sulphydryl (SH) groups of cysteine-containing proteins [22]resulting in a change in the oxidation state of protein constituents of the ion channel [23]. This suggestion is supported by the results of Chiamvimanvat et al. [24]which showed that sulphydryl oxidation of pore-forming subunits of the cloned rabbit smooth muscle L-type Ca++ channel resulted in irreversible reduction in ICa. The mechanism(s) by which adenosine interacts with ·O2– production is not known. Nevertheless, there is evidence in the literature that adenosine, acting via adenosine A1 receptors, does stimulate protein kinase C and regulates phospholipid metabolism [25, 26]and that both protein kinase C and phospholipids are potent modulators of superoxide generation [27, 28].

The possibility that NO does participate in the inhibitory action on the calcium current of adenosine when in combination with superoxide-generating agents cannot be completely excluded. NO is a target for ·O2– because of the high rate constant for reaction in aqueous solution. NO is known to react with ·O2–, resulting initially in the formation of peroxynitrite which also causes oxidation of sulphydrils [29]. However, since L-NAME did not modify the inhibition in ICa by superoxide-generating agents and adenosine, NO is not likely to play a major role in this mechanism.

Methylene blue has been used in many studies as an inhibitor of NO-sensitive guanylyl cyclase [8, 11, 17]since the superoxide it generates can scavenge NO leading to an inhibition of the enzyme. Mery et al. [8]have reported findings similar to our data that high concentrations of methylene blue (50 µM) by itself inhibited isoprenaline-stimulated ICa. Han et al. [11]also reported a non-specific effect of methylene blue on both calcium and potassium currents. Lower concentrations of methylene blue (5–10 µM), however, have been shown to inhibit the antiadrenergic effect on ICa of muscarinic agonists in rabbit SA nodal cells [11]and in guinea-pig ventricular myocytes [16]. This is not in accord with our data, but it is possible that muscarinic stimulation and adenosine produce different amounts of NO and, therefore, differentially scavenge the superoxide also produced. Muscarinic stimulation may produce sufficient NO to scavenge the superoxide generated by methylene blue whereas this may not be the case with adenosine. Further experiments are needed to investigate the observed differences between adenosine and muscarinic agonist-mediated effects.

The evidence that adenosine may both activate a NO-cGMP pathway and, in different situations, interact with superoxide radical production may have implications for the effect of adenosine in areas of the heart other than the A-V node. Under physiological conditions, tissues have adequate anti-oxidant defences such that free radicals are not present in a sufficiently high concentration to produce a biological effect [30]. Under conditions of myocardial ischaemia and reperfusion, however, it has been demonstrated that free radicals are produced [31]. The concentrations of purine and xanthine oxidase that were used in the present study are within the range of those used in other studies to demonstrate free-radical-induced changes in cardiac function [32]. Thus our results which suggested that there may be a free-radical-mediated effect of adenosine will only be of importance under pathophysiological conditions. Endogenous adenosine, NO and ·O2– are all candidates for the protective effect of ischaemic preconditioning [33, 34]and to our knowledge these are the first data which suggest an interrelationship between them. It is possible that in ventricular myocytes, under conditions of ischaemia and reperfusion, adenosine may also increase the production of NO and ·O2– both of which are capable of blocking Ca2+ channels. Block of ICa under ischaemic conditions may be cardioprotective because of the consequent reduction in calcium overload. Experiments should, therefore, be carried out in other cardiac cell types to establish whether or not adenosine's effects are mediated via similar signal transduction mechanisms.

In conclusion, we have demonstrated that adenosine can modulate isoprenaline-stimulated ICa in rabbit A-V nodal cells by both NO-cGMP and superoxide-dependent pathways. Further investigations are needed to clarify the interrelationship between adenosine, NO and ·O2– in A-V nodal and other cardiac cell types.

Time for primary review 17 days.


   Acknowledgements
 
The work was supported by the British Heart Foundation.


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

  1. Belardinelli L, Giles WR, West A. Ionic mechanisms of adenosine actions in pacemaker cells from rabbit heart. J Physiol 1988;405:615–633.
  2. Wang D, Belardinelli L. Mechanism of the negative inotropic effect of adenosine in guinea pig atrial myocytes. Am J Physiol 1994;267:H2420–H2429.
  3. Clemo HF, Belardinelli L. Effect of adenosine on atrioventricular conduction. I. Site and characterisation of adenosine action in the guinea pig atrioventricular node. Circ Res 1986;59:427–436.
  4. Rankin AC, Brooks R, Ruskin JN, McGovern BA. Adenosine and the treatment of supraventricular tachycardia. Am J Med 1991;92:655–664.
  5. Wesley RC, Lerman BB, DiMarco JP, Berne RM, Belardinelli L. Mechanism of atropine-resistant atrioventricular block during inferior myocardial infarction: Possible role of adenosine. J Am Coll Cardiol 1986;8:1232–1234.
  6. Martynyuk AE, Kane KA, Cobbe SM, Rankin AC. Adenosine increases potassium conductance in isolated rabbit atrioventricular nodal myocytes. Cardiovasc Res 1995;30:668–675.
  7. Martynyuk AE, Kane KA, Cobbe SM, Rankin AC. Nitric oxide mediates the anti-adrenergic effect of adenosine on calcium current in isolated rabbit atrio-ventricular nodal cells. Pflügers Arch – Eur J Physiol 1996;431:452–457.
  8. Mery PF, Pavoine C, Belhassen L, Pecker F, Fischmeister R. Nitric oxide regulates cardiac Ca2+ current. Involvement of cGMP-inhibited and cGMP-stimulated phosphodiesterases through guanylyl cyclase activation. J Biol Chem 1993;268:26286–26295.
  9. Wahler GM, Dollinger SJ. Nitric oxide donor SIN-1 inhibits mammalian cardiac calcium current through cGMP-dependent protein kinase. Am J Physiol 1995;268:C45–C54.
  10. Kirstein M, Rivet-Bastide M, Hatem S, Benardeau A, Mercadier JJ, Fischmeister R. Nitric oxide regulates the calcium current in isolated human atrial myocytes. J Clin Invest 1995;95:794–802.
  11. Han X, Shimoni Y, Giles WR. A cellular mechanism for nitric oxide-mediated cholinergic control of mammalian heart rate. J Gen Physiol 1995;106:45–65.
  12. Geiger J, Nolte C, Butt E, Sage SO, Walter U. Role of cGMP and cGMP-dependent protein kinase in nitrovasodilator inhibition of agonist-evoked calcium elevation in human platelets. Proc Natl Acad Sci USA 1992;89:1031–1035.
  13. Lincoln TM, Cornwell TL. Intracellular cyclic GMP receptor proteins. Fed Am Soc Exp Biol J. 1993;7:328–338.
  14. Beltman J, Becker DE, Butt E, et al. Characterization of cyclic nucleotide phosphodiesterases with cyclic GMP analogs: Topology of the catalytic domains. Mol Pharmacol 1995;47:330–339.
  15. Kontos HA, Wei EP, Mayhan WG. Hydroxyl radical-dependent inactivation of guanylate cyclase in cerebral arterioles by methylene blue and by LY83583. Stroke 1993;24:427–434.
  16. Marczin N, Ryan US, Catravas JD. Methylene blue inhibits nitrovasodilator- and endothelium-derived relaxing factor-induced cyclic GMP accumulation in cultured pulmonary arterial smooth muscle cells via generation of superoxide anion. J Pharmacol Exp Ther 1992;263:170–179.
  17. Levi RC, Alloatti G, Penna C, Gallo MP. Guanylate-cyclase-mediated inhibition of cardiac ICa by carbachol and sodium nitroprusside. Pflügers Arch 1994;426: 419–426.
  18. Han X, Kobzik L, Balligand J-L, Kelly RA, Smith TW. Nitric oxide synthase (NOS3)-mediated cholinergic modulation of calcium current in adult rabbit atrioventricular nodal cells. Circ Res 1996;78:998–1008.
  19. Baldwin DA, Jenny ER, Aisen P. The effect of human serum transferrin and milk lactoferrin on hydroxyl radical formation from superoxide and hydrogen peroxide. J Biol Chem 1984;259:13391–13394.
  20. Kuppusamy P, Zweier JL. Characterization of free radical generation by xanthine oxidase. Evidence for hydroxyl radical generation. J Biol Chem 1989;264:9880–9884.
  21. Fridovich I. Quantitative aspects of the production of superoxide anion radical by milk xanthine oxidase. J Biol Chem 1970;245:4053–4057.
  22. Means GE, Feeney RE. Chemical modification of proteins. San Francisco, CA: Holden-Day, 1971.
  23. Ruppersberg JP, Stocker M, Pongs O, Heinemann SH, Frank R, Koenen M. Regulation of fast inactivation of cloned mammalian IK(A) channels by cysteine oxidation. Nature 1991;352:711–714.
  24. Chiamvimonvat N, Orourke B, Kamp TJ, et al. Functional consequences of sulfhydryl modification in the pore-forming subunits of cardiovascular Ca2+ and Na+ channels. Circ Res 1995;76:325–334.
  25. Eckert R, Utz J, Trautwein W. Involvement of intracellular Ca2+ release mechanism in adenosine-induced cardiac Ca2+ current inhibition. Surgery 1993;114:334–342.
  26. Marala RB, Mustafa J. Adenosine A1 receptor-induced upregulation of protein kinase C: role of pertussis toxin-sensitive G protein(s). Am J Physiol 1995;269:H1619–H1624.
  27. Mayer AMS, Brenic S, Stocker R, Glaser KB. Modulation of superoxide generation in in vivo lipopolysaccharide-primed rat alveolar macrophages by arachidonic acid and inhibitors of protein kinase C, phospholipase A2, protein serine–threonine phosphatase(s), protein tyrosine kinase(s) and phosphatase(s). J Pharmacol Exp Ther 1995;274:427–436.
  28. Mayer AMS, Spitzer JA. Modulation of superoxide generation in in vivo lipopolysaccharide-primed Kupffer cells by staurosporine, okadaic acid, manoalide, arachidonic acid, genistain and sodium ortovanadate. J Pharmacol Exp Ther 1993;268:238–247.
  29. Radi R, Beckman JS, Bush KM, Freeman BA. Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Arch Biochem Biophys 1991;288:481–587.
  30. Rice Evans C, Burdon R. Free radical–lipid interactions and their pathological consequences. Prog Lipid Res 1993;32:71–110.
  31. Zhou X, Zhai X, Ashraf M. Direct evidence that initial oxidative stress triggered by preconditioning contributes to second window of protection by endogenous antioxidant enzyme in myocytes. Circulation 1996;93:1177–1184.
  32. Burton KP, McCord JM, Geetha G. Myocardial alterations due to free-radical generation. Am J Physiol 1984;246:H776–H783.
  33. Chen W, Gabel S, Steenbergen C, Murphy E. A redox-based mechanism for cardioprotection induced by ischemic preconditioning in perfused rat heart. Circ Res 1995;77:424–429.
  34. Liu GS, Thronton J, Van Winkle DM, et al. Protection against infarction afforded by preconditioning is mediated by A1 adenosine receptors in rabbit heart. Circulation 1991;84:350–356.

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Physiol.Home page
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]

Home page
 J Biol RhythmsHome page
I. V. Zhdanova, M. Simmons, J. N. Marcus, A. C. Busza, O. U. Leclair, and J. A. Taylor
Nocturnal Increase in Plasma cGMP Levels in Humans
J Biol Rhythms, August 1, 1999; 14(4): 307 - 313.
[Abstract] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Martynyuk, A.E
Right arrow Articles by Rankin, A.C
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Martynyuk, A.E
Right arrow Articles by Rankin, A.C
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?