Skip Navigation

Cardiovascular Research 2000 47(1):81-89; doi:10.1016/S0008-6363(00)00057-2
© 2000 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
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
Google Scholar
Right arrow Articles by Mohan, R. M
Right arrow Articles by Paterson, D. J
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mohan, R. M
Right arrow Articles by Paterson, D. J
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2000, European Society of Cardiology

Activation of sulphonylurea-sensitive channels and the NO-cGMP pathway decreases the heart rate response to sympathetic nerve stimulation

Ravi M Mohan* and David J Paterson

University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK

* Corresponding author. Tel.: +44-1865-272-481 ravi.mohan{at}physiol.ox.ac.uk david.paterson{at}physiol.ox.ac.uk

Received 8 December 1999; accepted 15 February 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: Activation of ATP sensitive K+ channels (KATP) and the NO-cGMP pathway have both been implicated in reducing norepinephrine (NE) release from cardiac sympathetic nerves during stimulation. Our aim was to test whether these pathways could interact and modulate cardiac excitability during sympathetic nerve stimulation (SNS). Methods: The effect of inhibitors and activators of KATP channels and the NO-cGMP pathway on the heart rate (HR) response to cardiac SNS in the isolated guinea pig (Cavia porcellus) double atrial/right stellate ganglion preparation was studied (n=48). Results: The KATP channel activator, diazoxide (100 µM, n=6) or hypoxia (0% O2/5% CO2, n=6) significantly attenuated the HR response to 3 Hz SNS by –10±4% and –27±6% respectively; an effect that was reversed by the KATP channel inhibitor, glibenclamide (30 µM). Glibenclamide (n=6) on its own enhanced the HR response to SNS by 20±8%. Bath applied NE (0.1–0.7 µM, n=6) did not affect the HR response to diazoxide, although an increased response to glibenclamide was observed at 0.3 and 0.5 µM NE. In the presence of 8-Br-cGMP (0.5 mM, n=7), diazoxide further decreased the HR response SNS (19±3%). The NO synthase inhibitor, N-{omega}-nitro-L-arginine (100 µM) significantly increased the HR response (13±3%) to SNS in the presence of diazoxide (100 µM, n=6). This effect was reversed with excess (1 mM) L-arginine. Conversely, the NO donor, sodium nitroprusside (SNP, 20–100 µM) significantly attenuated the HR response to SNS. The addition of glibenclamide (30 µM, n=10) could still enhance the HR response (42±15%) to SNS. Similar results were seen with the cyclic GMP analogue, 8-Br-cGMP (0.5 mM, n=12). Conclusions: Our results indicate that NO and sulphonlyurea-sensitive channels act in a complementary fashion, but appear to be independent of each other in the regulation of HR during cardiac SNS activation.

KEYWORDS Nitric oxide; K-ATP channel; Heart rate (variability); Hypoxia/anoxia; Autonomic nervous system


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Depolarization of the presynaptic sympathetic nerve terminal leads to an influx of calcium and exocytotic release of norepinephrine (NE). Once released into the synaptic cleft, NE can influence subsequent transmitter release via activation of presynaptic {alpha}2-adrenoreceptor modulatory pathways (autoinhibition) [7,11]. Similarly, activation of presynaptic muscarinic M3-receptors [15,18], A1 adenosine receptors [7,24] and neuronally generated nitric oxide [31] are all able to inhibit NE release from sympathetic nerves in the heart.

More recently, activated neuronal ATP-sensitive potassium channels (KATP) have been implicated as presynaptic inhibitors of stimulation-evoked NE release in the guinea pig atria [26]. Activation of KATP channels either by KATP channel openers or by depletion of ATP has been proposed as the mechanism by which hyperpolarization of the nerve terminal and shortening of action potential duration reduces calcium influx and exocytotic release of NE [34]. Similarly, there is evidence that nitric oxide (NO), like KATP activation, inhibits peripheral sympathetic activity in the heart. Inhibition of endogenous NO production with non-isoform specific and neuronal NOS (nNOS) inhibitors increases NE release during cardiac sympathetic nerve stimulation (SNS) [31]. However, while the idea of a potential synergistic relationship between KATP channels and NO as modulators of NE release is attractive, the response of these pathways on organ and tissue behaviour is poorly understood.

The interaction between NO and KATP channels was first proposed by Garland and co-workers [8] who demonstrated that the NO-induced hyperpolarisation in rat mesentery was abolished by KATP inhibition. In contrast, more recent studies have shown that both NO-induced hyperpolarisation [37] and vasodilation [12,33] are not mediated by activation of KATP channels. Whether these channels play a functionally significant role in the sympathetic control of cardiac excitability and whether they are modulated by the NO-cGMP pathway is not known.

Therefore, the aims of this study were two-fold. First, to investigate whether modulators of sulphonylurea-sensitive channels (KATP channels) alter the peripheral sympathetic control of heart rate in the isolated guinea-pig atria. Secondly, to determine whether an interaction between NO and KATP channels affects the HR response to peripheral sympathetic activation.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Animal care
All experiments conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985) and were performed in accordance with Home Office license requirements (PPL 30/1133, Queen Anne's Gate, London, UK) and the Animals (Scientific Procedures) Act 1986 (UK). Forty eight male guinea pigs (Harlan, UK) initially weighing between 250 and 350 g were housed in a temperature controlled room (20±1°C) with a 12-h light/dark cycle. Chow and water were provided ad libitum.

2.2 Guinea pig sino atrial node/right stellate ganglion preparation
Animals were killed by cervical dislocation followed by exsanguination. The thorax and mediastinum were rapidly removed and placed in Tyrode's solution aerated with 95% O2/5% CO2. The ventricles were perfused with heparinised Tyrode's solution (1000 U ml–1) and excised. The atria and right stellate ganglion were dissected free and sutures (Ethicon 6/0 silk) were placed on the lateral edges of both atria. The preparation was then transferred to a preheated (37±0.1°C) water-jacketed bath containing 100 ml of oxygenated Tyrode's solution. The atria were vertically mounted with the suture in the left atrium connected to a stainless steel hook, and the suture in the right atrium attached to an isometric force transducer (Harvard Apparatus, Model # 60-2997, MA, USA). The right stellate ganglion was placed through a pair of platinum ring electrodes connected to a stimulator. Data were collected on a Power Macintosh 7500 computer (Apple Systems, CA, USA) using a Biopac MP100 acquisition system and Acknowledge software (Biopac Systems Inc. CA, USA). Heart Rate (bpm) was triggered from contraction, and signals were displayed in real time. Data were stored on CD Rom for offline analysis.

2.3 Solutions and drugs
Tyrode's solution contained (mM) NaCl 120, KCl 4, MgCl2 2, NaH2PO4 0.1, NaHCO3 25, CaCl2 2, and glucose 11. The solution was aerated with 95%O2/5%CO2 (pH=7.4) and its temperature maintained at 37±0.1°C (Grant Instruments, Cambridge, UK).

KATP channel modulators, glibenclamide, tolbutamide and diazoxide were added from stock solutions 100 mM (in DMSO), 10 mM (in DMSO) and 50 mM (in 0.1 M NaOH), respectively. Solutions of cesium chloride (CsCl), sodium nitroprusside (SNP), N-{omega}-nitro-L-arginine (L-NA) and L-arginine (L-arg) were all dissolved in Tyrode's solution and prepared immediately prior to application. Norepinephrine bitartrate (NE), and the membrane permeable cGMP analogue 8-Br-cGMP were added from 1 mM and 0.1 M stock solutions respectively, made up prior to the experiment using reagant grade water from an Elga water purification system. All other chemicals were purchased from Sigma Aldrich, Dorset, UK.

2.4 Experimental protocols
The atria equilibrated in Tyrode's solution for 45–90 min until the heart rate did not alter by 10 bpm over a 20 min period. Following this period, the stellate ganglion was stimulated at 3 Hz, 10 V, 1 ms pulse width for 30 s at 2–3 min intervals. Experimental protocols commenced after three consistent positive chronotropic responses to ganglion stimulation were achieved. Prior to each pharmacological intervention, the ganglion was stimulated at 1, 3 and 5 Hz (10 V, 1 ms). Fresh Tyrode's solution was then placed in the organ bath prior to incubation with either a modulator of the NO-cGMP pathway or KATP channels. Following this, a second period of 1, 3 and 5 Hz sympathetic stimulation was completed. A second incubation period with a combination of NO-cGMP pathway and KATP channel modulators preceded the final cycle of SNS. The change in heart rate with SNS was calculated by difference in 5-s averages in heart rate taken prior to the onset and cessation of a stimulation period.

2.4.1 Effect of KATP channel activation on the HR response to SNS
We evaluated the effects of the KATP opener, diazoxide (5–100 µM) on the increase in heart rate with SNS. Diazoxide was used as the primary pharmacological KATP activator since it was shown to be a more potent inhibitor of NE release than both cromokalim and pinacidil, the latter of which actually enhances adrenergic neurotransmission [26,34]. Concentrations of diazoxide (5, 10, 50 and 100 µM) were added cumulatively and allowed to incubate for 6 min each prior to SNS. To antagonize the effects of the KATP opener, glibenclamide (30 µM) was added 10 min prior to a single 100 µM addition of diazoxide.

The HR response to SNS was measured following activation KATP channels by hypoxia. A control 3 Hz stimulation preceded a 6 min in-vitro bout of hypoxia (0% O2/5% CO2). During the last 30 s of the in-vitro hypoxic episode, the right stellate ganglion was stimulated at 3 Hz. A second bout of in-vitro hypoxia was introduced after the addition of glibenclamide (30 µM; 10 min incubation) and a third stimulation of the right stellate ganglion during this hypoxic period was completed.

2.4.2 Effect of KATP inhibition on HR response to SNS
We evaluated the effects of the KATP inhibitors, glibenclamide (5–30 µM) and tolbutamide (1–5 µM) on the increase in heart rate with SNS. Glibenclamide (5 µM) or tolbutamide (1 µM) was added to the organ bath and allowed 10–12 min incubation prior to SNS. Subsequent concentrations of glibenclamide (10 and 30 µM) or tolbutamide (2 and 5 µM) were added cumulatively at 10–12 min intervals each prior to SNS.

2.4.3 Effect of diazoxide and glibenclamide on the increase in heart rate with bath-applied NE
The increase in heart rate with exogenous NE (cumulative addition: 0.1–0.7 µM) was measured in the presence of single doses of diazoxide (5, 10, 50 and 100 µM) or glibenclamide (5, 10, and 30 µM) to determine whether modulation of KATP channels altered the HR response to SNS via a pre- or post-synaptic mechanism. The Tyrode's solution was replaced in the organ bath prior to the addition of each concentration of diazoxide or glibenclamide.

2.4.4 Effect of dual activation of NO-cGMP pathway and KATP channels on the HR response to SNS
We simultaneously activated both the NO-cGMP pathway and KATP channels to define whether they acted independently to modulate the HR response to SNS. After the first cycle of SNS in the presence of CsCl (2 mM, 12 min equilibration) to minimise the changes in baseline heart rate following activation of the NO-cGMP pathway [23], with the membrane permeable cGMP analogue, 8-Br-cGMP (0.5 mM, 15 min incubation) was added and a second cycle of SNS was repeated. The final cycle of SNS was completed after addition of diazoxide (100 µM).

2.4.5 Role of NOS inhibition on the heart rate response to SNS with KATP activation
We tested the effects of the NOS inhibitor, N-{omega}-nitro-L-arginine (L-NA, 100 µM) in the presence of the KATP opener, diazoxide (100 µM) on the increase in heart rate with SNS. Control stimulations were performed in the presence of diazoxide (6 min incubation). For the second cycle of SNS, fresh Tyrode's solution was then placed in the organ bath and L-NA (100 µM) was equilibrated with the tissue for 20 min prior to the addition of diazoxide (100 µM). Following the addition of fresh Tyrode's solution, L-NA (100 µM) and L-arginine (1 mM) (20–25 min incubation), diazoxide (100 µM) was added and the final cycle of SNS was completed. The increases in heart rate with SNS were averaged for diazoxide only, L-NA+ diazoxide and L-NA+L-arginine+diazoxide.

2.4.6 Role of KATP inhibition on the heart rate response to SNS with NO-cGMP activation
We examined the role of glibenclamide (30 µM) in the presence of the NO donor SNP or 8-Br-cGMP, on the increase in heart rate with SNS. The right stellate ganglion was stimulated during a control period prior to addition of SNP (100 µM, 15 min incubation) or 8-Br-cGMP (0.5 mM, 15 min incubation; in the presence of 2 mM CsCl to minimise the change in baseline heart rate caused by 8-Br-cGMP) and again following this equilibration period. Glibenclamide (30 µM; 10 min incubation) was added to the organ bath in the presence of the equilibrated SNP or 8-Br-cGMP and the stellate ganglion was stimulated for a third cycle. The above NO donor protocol was repeated using a lower concentration of SNP (20 µM) in the presence of CsCl (2 mM, 12 min incubation).

2.5 Statistical analysis
Data are presented as the mean±SEM unless otherwise specified. For the double atrial/right stellate ganglion preparations, one-way repeated measures ANOVA followed by a Student Newman-Keuls test for pairwise comparison was used to evaluate intra- and inter-group differences in the positive chronotropic response to SNS at 1, 3 and 5 Hz. Statistical significance was accepted at P≤0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
After 90–120 min of equilibration, the spontaneous beating rate of the atrial preparations averaged 184± 3 bpm (n=48).

3.1 Effect of KATP modulators on the increase in heart rate with SNS
Fig. 1 shows a representative raw data trace for the effect of sympathetic stimulation (1, 3 and 5 Hz) on heart rate (bpm) in isolated atrial preparations in the presence of the KATP channel activator, diazoxide (100 µM, n=6). Diazoxide significantly attenuated the HR response to SNS (P<=0.05). This response was antagonized by the KATP channel inhibitor, glibenclamide (see Table 1). No effect on baseline heart rate was observed with diazoxide (5–100 µM).


Figure 1
View larger version (9K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Representative raw data traces showing the effects of cardiac SNS (1, 3 and 5 Hz, 10 v, 1 ms pulse width) on heart rate (bpm) in a double atrial/right stellate ganglion preparation under control conditions (top trace) and in the presence of the KATP opener, diazoxide (100 µM; lower trace). The right stellate ganglion was stimulated for 30 s, with 90–120 s between each stimulation period. The time calibration bar refers to both traces.

 

View this table:
[in this window]
[in a new window]

  		Table 1 Effect of KATP activators and inhibitors on the heart rate response to SNSa

 
A period of in-vitro hypoxia (0% O2/5% CO2) was introduced to examine the effects of hypoxic activation of KATP channels on the HR response to SNS. After approximately 6 min of hypoxia, the HR response to 3 Hz SNS was significantly reduced (P<0.05; n=6, Fig. 2). This effect was reversed by the addition of glibenclamide to the atrial preparation prior to a subsequent episode of hypoxia.


Figure 2
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 The effect in-vitro hypoxia (0%O2/5%CO2) and its reversal with glibenclamide (30 µM) on the increase in heart rate with right stellate ganglion stimulation (10 V, 1 ms pulse width, 30 s duration) at 3 Hz (n=6). Hypoxia significantly decreased the magnitude of the positive chronotropic response to sympathetic activation and this effect was significantly enhanced with glibenclamide (*, P<0.05; ANOVA).

 
Glibenclamide (n=6) enhanced the HR response to SNS in a concentration dependent manner, reaching a maximal response at 30 µM (Fig. 3, Table 1). Baseline HR significantly declined from 165±7 bpm during control conditions to 151±7 bpm (P<0.05) in the presence of 30 µM glibenclamide. Tolbutamide (n=5) enhanced the HR response to SNS at 3 and 5 Hz in a similar manner to glibenclamide (Table 1), however, no significant changes in baseline heart rate were observed.


Figure 3
View larger version (10K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Representative raw data traces showing the effects of cardiac SNS (1, 3 and 5 Hz, 10 V, 1 ms pulse width) on heart rate (bpm) in a double atrial/right stellate ganglion preparation under control conditions (top trace) and in the presence of the KATP inhibitor, glibenclamide 30 µM; lower trace). The right stellate ganglion was stimulated for 30 s, with 90–120 s between each stimulation period. The time calibration bar refers to both traces.

 
3.2 Effect of KATP modulators on the increase in heart rate with bath-applied norepinephrine
The positive chronotropic effect to the cumulative addition of NE (0.1–0.7 µM) was not significantly different between control and diazoxide (5–100 µM, n=6), although, 30 µM glibenclamide significantly enhanced (P<0.05) the chronotropic effect of NE at 0.3 and 0.5 µM (n=6, Table 2)


View this table:
[in this window]
[in a new window]

  		Table 2 Effect of KATP activators and inhibitors on the heart rate response to SNSa

 
3.3 Effect of activation of both the NO-cGMP pathway and KATP channels on the HR response to SNS
8-Br-cGMP significantly reduced the HR response 1, 3 and 5 Hz (P<0.05) nerve stimulation in the presence of 2 mM CsCl (n=7). This effect was then further significantly reduced (P<0.05) in the presence of diazoxide at 3 and 5 Hz stimulation (Fig. 4). All effects were fully reversible on wash-off, confirming the preparations were not run down.


Figure 4
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 The effect NO-cGMP pathway activation with 8-Br-cGMP (0.5 mM) and the combined effect of 8-Br-cGMP and Diazoxide (100 µM) on the increase in heart rate with right stellate ganglion stimulation (10 V, 1 ms pulse width, 30 s duration) at 3 Hz (n=7). 8-Br-cGMP significantly reduced the HR response to SNS (*, P<0.05; ANOVA) and this effect was then further significantly reduced (**, P<0.05; ANOVA) in the presence of diazoxide. All effects were fully reversible on wash-off, confirming the preparations were not run down.

 
3.4 Effect of NO modulators on KATP mediated heart rate response to SNS
L-NA enhanced the HR response to SNS (n=5; change in HR for Control, L-NA, L-NA+L-arginine 35±2, 47±3, 35±2; 86±5, 88±6, 77±4 and 98±5, 111±4, 98±7 at 1 Hz, 3 Hz and 5 Hz respectively). This response was greater than with diazoxide on its own at all frequencies, however only reached statistical significance at 1 Hz. The HR response to SNS with diazoxide was significantly enhanced (P<0.05) in the presence of the non-isoform selective NOS inhibitor L-NA (n=6) at all frequencies of nerve stimulation (Fig. 5). The effect of L-NA was reversed with excess L-arginine (100 µM L-NA/100 µM Diazoxide+1 mM L-arginine) at 1 and 5 Hz SNS.


Figure 5
View larger version (8K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 The effect NOS inhibition with L-NA (100 µM) in presence of diazoxide (100 µM) and its reversal with L-arginine (1 mM) on the increase in heart rate with right stellate ganglion stimulation (10 V, 1 ms pulse width, 30 s duration) at 1, 3 and 5 Hz. L-NA enhanced the HR response to SNS and this response was greater than with diazoxide only (n=5; not shown). This trend was observed at all frequencies, however only reached statistical significance at 1 Hz ({dagger}, P<0.05; ANOVA). The HR response to SNS with diazoxide was signficianc enhanced (*, P<0.05; ANOVA) in the presence of L-NA at all frequencies of nerve stimulation. The effect of L-NA was reversed with excess L-arginine (100 µM L-NA/100 µM Diazoxide+1 mM L-arginine) at 1 and 5 Hz SNS (**, P<0.05; ANOVA).

 
Conversely, the NO donor, SNP, significantly attenuated (P<0.05) the HR response to SNS, whereas addition of glibenclamide had the opposite effect (n=6, Table 3). To verify that the effect of KATP inhibition on the SNP mediated HR response to SNS was not due to alterations in baseline heart rate, experiments were repeated using a lower concentration of SNP (20 µM) in the presence of CsCl. Fig. 6a (top) shows the mean HR response to SNS with SNP (20 µM) and glibenclamide (n=10). Glibenclamide enhanced the HR response to SNS in the presence of SNP. Similarly, glibenclamide could still increase the HR response to SNS when the response had been significantly reduced (P<0.05) at 1, 3 and 5 Hz nerve stimulation with 8-Br-cGMP (in the presence of 2 mM CsCl; n=13, Fig. 6b, Table 3)


View this table:
[in this window]
[in a new window]

  		Table 3 Effect of NO modulators on KATP-mediated heart rate response to SNSa

 

Figure 6
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 The effect of glibenclamide (30 µM) in the presence of the NO donor SNP (20 µM, n=10:A) or the cGMP analogue, 8-Br-cGMP (0.5 mM; n=12:B) on the increase in heart rate with SNS. Glibenclamide could still increase the HP response to SNS (**, P<0.05; ANOVA) when the response has been significantly reduced (*, P<0.05; ANOVA) at 3 Hz nerve stimulation with SNP and 8-Br-cGMP.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The new finding in this study is that sulphonylurea-sensitive channels (KATP channels) and the NO-cGMP pathway can significantly regulate the HR response to peripheral cardiac sympathetic nerve stimulation. In addition, activation of both the NO-cGMP pathway and KATP channels reduced the HR response to SNS further than activation of the NO-cGMP pathway alone, indicating that both pathways can act independently.

4.1 Effect of KATP modulators on the heart rate response to sympathetic nerve stimulation
Myocardial KATP channels are closed under normal physiological conditions, but they are activated by either a decrease in intracellular ATP concentration [25], hypoxia [5,39] or by specific KATP channel activators such as diazoxide [9], cromakalim or pinacidil [3,34]. Their activation during cardiac ischaemia postpones the onset of irreversible damage and reduces the size of myocardial infarct (for review see [17,30,36]). Subsequent to their localization in pre- and postsynaptic neurons [21], recent work has shown that KATP activation by cromokalim and diazoxide (100 µM) inhibits stimulation-evoked NE release in the guinea pig atria [26]. We provide functional support for this observation and show that similar concentrations of diazoxide decrease the HR response to SNS.

Studies using radiochemical binding to NE (3H-NE) similar to that described by Oe and co-workers [26] found that KATP activation with either cromokalim or diazoxide inhibited stimulation evoked 2-[14C] NE efflux from the canine/bovine mesenteric artery [3], as well as [3H] NE release from the dorsal metatarsal vein and rat neocortex [34]. Similarly, Remme and co-workers [29] found that cromokalim reduced stimulation evoked NE release in globally ischaemic rabbit hearts and Takata and co-workers [35] reported that 5 min hypoxia in rat cortical slices linearly reduces both tissue ATP content and evoked NE release, the latter effect being reversed by glibenclamide. In our study, the increase heart rate with cardiac SNS was attenuated both in the presence of diazoxide or during hypoxia and this was reversed by glibenclamide suggesting direct modulation of KATP channels.

It is possible that glibenclamide could be acting on other ionic conductances involved in transmitter release since this sulphonylurea has been shown to inhibit Ca2+ activated K+ channels in neuroblastoma cell lines [28]. However, inhibition of KATP channels in human pancreatic β-cells with tolbutamide (10 mM) [2] or in skeletal muscle with glibenclamide (up to 100 µM) has been shown to be ineffective in blocking either Ca2+ activated K+ channels or voltage gated K+ channels [16]. In our study, the concentration of both glibenclamide and tolbutamide were relatively low compared to concentrations needed to inhibit KATP channels, and they were similar to others who have directly modulated transmitter release with these drugs [26,34]. Nevertheless, we cannot completely exclude the possibility that other ionic conductances were unaffected by glibenclamide since Oe and co-workers [26] reported that the relatively specific KATP blocker 5-hydroxydecanoate (5-HD) was ineffective in reducing stimulated induced NE release.

In the absence of either pharmacological or physiological KATP channel activation, application of sulphonylureas to rat substantia nigra slices induces release of transmitter [1] suggesting that activated KATP channels may play a role in the regulation of transmitter release under normal conditions [19,38]. In the present study, both sulphonylureas, tolbutamide and glibenclamide, significantly enhanced the HR response to 3 Hz SNS by approximately 17% and 25%, respectively. We found that glibenclamide enhanced the HR response to bath applied NE, however, diazoxide did not have any appreciable effect. Taken together, the changes in the HR response to either SNS or exogenous NE elicited by the KATP inhibition were greater than those seen with KATP activation. These findings are in agreement with recent studies suggesting that KATP channels may be partly activated in the isolated atrial preparation [26] and that under normal circumstances they are sensitive to glibenclamide and responsive to β-adrenergic stimulation [38].

The differences in the effect of diazoxide and glibenclamide on the HR response to bath applied NE may be due to their different specificity towards mitochondrial and sarcolemmal KATP channels. Both types of channel are inhibited by glibenclamide [27], however, diazoxide has a much higher affinity for the mitochondrial KATP channel [10]. Therefore, it is likely that the enhanced response to bath applied NE in the presence of glibenclamide may occur as a result of sarcolemmal KATP activation.

4.2 Interaction between KATP and NO modulators
There is good evidence that NO, like KATP activation, inhibits peripheral sympathetic activity in the heart. Inhibition of endogenous NO production with non-isoform specific and neuronal NOS (nNOS) inhibitors increases both NE release during cardiac SNS [31] and the heart rate response to SNS [6,32]. Similarly, nNOS inhibition has been shown to enhance, whereas an NO donor or cGMP analogue attenuates, the heart rate and inotropic responses to SNS in the isolated guinea-pig atria [4]. The signalling pathway responsible for NO inhibiting sympathetic neurotransmission is not completely understood, but it could involve cGMP activation of protein kinase G [20] and phosphodiesterases that decrease cAMP-dependent phosphorylation of neuronal Ca2+ channels.

Recently, Murphy and co-workers [22] reported that in rabbit vascular smooth muscle, hyperpolarisation elicited by an NO donor was glibenclamide sensitive and suggested that there was activation of KATP channels via a cGMP mediated pathway. Similarly, it has been shown in guinea pig mesentery that sodium nitroprusside-induced hyperpolarisation was reduced by glibenclamide [38]. In contrast to these studies, hyperpolarisation-induced by either endogenous or exogenous NO in the rat aorta [37] or SNP/cGMP-induced vasodilation in porcine arterioles [12] has been shown to be independent of KATP activation. We also find no evidence to suggest that KATP channels and the NO-cGMP pathway are interdependent since we observed a further reduction in the HR response to SNS with KATP opening in the presence of NO-cGMP activation.

The present study provides functional support for the role of KATP channels and the NO-cGMP pathway in the presynaptic modulation of cardiac excitability. These pathways appear to be distinct and regulated by different processes and may contribute, with autoinhibition, to the regulation of neurotransmission and post junctional behaviour. The role of these pathways under normal physiological conditions is not established, although in pathophysiological states, they may play a dominant role in modulating the response to sympathetic activation. In particular, conditions that promote KATP activation (i.e. ischaemia) and NO production (i.e. sepsis, heart failure) may reduce local exocytotic NE release to protect the myocardium by reducing HR to minimise oxygen consumption and cardiac work. This response will have to compete with sympatho-sympathetic reflexes increasing NE release to an ischaemic area. Similarly, the NO-cGMP pathway may directly activate cardiac pacemaking [13,14] as well as reduce NE release [31]. The net effect of these pathways on cardiac excitability is not known. Whether they could provide sites for therapeutic targets to minimise the effects of high sympathetic drive remains to be established. However, any therapeutic use in-vivo would have to counter the effects of sympatho-vagal reflexes activated by the fall in blood pressure caused by the NO-cGMP pathway and KATP channel activation on vascular smooth muscle.

Time for primary review 21 days.


   Acknowledgements
 
We are grateful to the British Heart Foundation for supporting this study. RM is supported by a Heart and Stroke of Canada Junior Research Traineeship and an NSERC PGS-B Studentship (Canada).


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

  1. Amoroso S., Schmid-Antomarchi H., Fosset M., Lazdunski M. Glucose, sulfonylureas, and neurotransmitter release: role of ATP-sensitive K+ channels. Science (1990) 247:852–854.[Abstract/Free Full Text]
  2. Ashcroft F.M., Kakei M., Gibson J.S., Gray D.W., Sutton R. The ATP- and tolbutamide-sensitivity of the ATP-sensitive K-channel from human pancreatic B cells. Diabetologia (1989) 32:591–598.[ISI][Medline]
  3. Cai B., Hao Q., Greenberg S.S., et al. Differential effects of pinacidil and cromakalim on vascular relaxation and sympathetic neurotransmission. Can J Physiol Pharmacol (1994) 72:801–810.[ISI][Medline]
  4. Choate J.K., Paterson D.J. Nitric oxide inhibits the positive chronotropic and inotropic responses to sympathetic nerve stimulation in the isolated guinea-pig atria. J Auton Nerv Syst (1999) 75:100–108.[CrossRef][ISI][Medline]
  5. Deutsch N., Klitzner T.S., Lamp S.T., Weiss J.N. Activation of cardiac ATP-sensitive K+ current during hypoxia: correlation with tissue ATP levels. Am J Physiol (1991) 261:H671–H676.[ISI][Medline]
  6. Elvan A., Rubart M., Zipes D.P. NO modulates autonomic effects on sinus discharge rate and AV nodal conduction in open-chest dogs. Am J Physiol (1997) 272:H263–H271.[ISI][Medline]
  7. Fuder H. Selected aspects of presynaptic modulation of noradrenaline release from the heart. J Cardiovasc Pharmacol (1985) 7(Suppl_5):S2–S7.[CrossRef]
  8. Garland J.G., McPherson G.A. Evidence that nitric oxide does not mediate the hyperpolarization and relaxation to acetylcholine in the rat small mesenteric artery. Br J Pharmacol (1992) 105:429–435.[ISI][Medline]
  9. Garlid K.D., Paucek P., Yarov-Yarovoy V., et al. Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K+ channels. Possible mechanism of cardioprotection. Circ Res (1997) 81:1072–1082.[Abstract/Free Full Text]
  10. Garlid K.D., Paucek P., Yarov-Yarovoy V., Sun X., Schindler P.A. The mitochondrial KATP channel as a receptor for potassium channel openers. J Biol Chem (1996) 271:8796–8799.[Abstract/Free Full Text]
  11. Hein L., Altman J.D., Kobilka B.K. Two functionally distinct alpha-2adrenergic receptors regulate sympathetic neurotransmission. Nature (1999) 402:181–183.[CrossRef][Medline]
  12. Hein T.W., Kuo L. cAMP-independent dilation of coronary arterioles to adenosine: role of nitric oxide. G proteins, and KATP channels. Circ Res (1999) 85:634–642.[Abstract/Free Full Text]
  13. Hogan N., Casadei B., Paterson D.J. Nitric oxide donors can increase heart rate independent of autonomic activation. J Appl Physiol (1999) 87:97–103.[Abstract/Free Full Text]
  14. Hogan N., Kardos A., Paterson D.J., Casadei B. Effect of exogenous nitric oxide on baroreflex function in humans. Am J Physiol (1999) 277:H221–H227.[ISI][Medline]
  15. Kobayashi O., Nagashima H., Duncalf D., et al. Direct evidence that pancuronium and gallamine enhance the release of norepinephrine from the atrial sympathetic nerve by inhibiting prejunctional muscarinic receptors. J Auton Nerv Syst (1987) 18:55–60.[CrossRef][ISI][Medline]
  16. Light P.E., French R.J. Glibenclamide selectively blocks ATP-sensitive K+ channels reconstituted from skeletal muscle. Eur J Pharmacol (1994) 259:219–222.[CrossRef][ISI][Medline]
  17. Liu Y., Sato T., Seharaseyon J., Szewczyk A., Marban E. Mitochondrial ATP-dependent potassium channels. Viable candidate effectors of ischemic preconditioning. Ann NY Acad Sci (1999) 874:27–37.[Abstract/Free Full Text]
  18. Manabe N., Foldes F.F., Torocsik A., Nagashima H., Goldiner P.L., Vizi E.S. Presynaptic interaction between vagal and sympathetic innervation in the heart: modulation of acetylcholine and noradrenaline release. J Auton Nerv Syst (1991) 32:233–242.[CrossRef][ISI][Medline]
  19. Meir A., Ginsburg S., Butkevich A., et al. Ion channels in presynaptic nerve terminals and control of transmitter release. Physiol Rev (1999) 79:1019–1088.[Abstract/Free Full Text]
  20. Mery P.F., Lohmann S.M., Walter U., Fischmeister R. Ca2+ current is regulated by cyclic GMP-dependent protein kinase in mammalian cardiac myocytes. Proc Natl Acad Sci USA (1991) 88:1197–1201.[Abstract/Free Full Text]
  21. Mourre C., Widmann C., Lazdunski M. Sulfonylurea binding sites associated with ATP-regulated K+ channels in the central nervous system: autoradiographic analysis of their distribution and ontogenesis, and of their localization in mutant mice cerebellum. Brain Res (1990) 519:29–43.[CrossRef][ISI][Medline]
  22. Murphy M.E., Brayden J.E. Nitric oxide hyperpolarizes rabbit mesenteric arteries via ATP-sensitive potassium channels. J Physiol (Lond) (1995) 486:47–58.[Abstract/Free Full Text]
  23. Musialek P., Lei M., Brown H.F., Paterson D.J., Casadei B. Nitric oxide can increase heart rate by stimulating the hyperpolarization-activated inward current I(f). Circ Res (1997) 81:60–68.[Abstract/Free Full Text]
  24. Nakatsuka H., Nagano O., Foldes F.F., Nagashima H., Vizi E.S. Effects of adenosine on norepinephrine and acetylcholine release from guinea pig right atrium: role of A1-receptors. Neurochem Int (1995) 27:345–353.[CrossRef][ISI][Medline]
  25. Noma A. ATP-regulated K+ channels in cardiac muscle. Nature (1983) 305:147–148.[CrossRef][Medline]
  26. Oe K., Sperlagh B., Santha E., et al. Modulation of norepinephrine release by ATP-dependent K(+)-channel activators and inhibitors in guinea-pig and human isolated right atrium. Cardiovasc Res (1999) 43:125–134.[Abstract/Free Full Text]
  27. Paucek P., Mironova G., Mahdi F., Beavis A.D., Woldegiorgis G., Garlid K.D. Reconstitution and partial purification of the glibenclamide-sensitive. ATP-dependent K+ channel from rat liver and beef heart mitochondria. J Biol Chem (1992) 267:26062–26069.[Abstract/Free Full Text]
  28. Reeve H.L., Vaughan P.F., Peers C. Glibenclamide inhibits a voltage-gated K+ current in the human neuroblastoma cell line SH-SY5Y. Neurosci Lett (1992) 135:37–40.[CrossRef][ISI][Medline]
  29. Remme C., Schumacher C., deJong J., Coronel R., Wilde A. Cromokalim reduces myocardial noradrenaline release during global ischaemia in rabbits. Eur Heart J (1998) 19(supplement):632.
  30. Schotborgh C.E., Wilde A.A. Sulfonylurea derivatives in cardiovascular research and in cardiovascular patients. Cardiovasc Res (1997) 34:73–80.[Abstract/Free Full Text]
  31. Schwarz P., Diem R., Dun N.J., Forstermann U. Endogenous and exogenous nitric oxide inhibits norepinephrine release from rat heart sympathetic nerves. Circ Res (1995) 77:841–848.[Abstract/Free Full Text]
  32. Sears C.E., Choate J.K., Paterson D.J. Effect of nitric oxide synthase inhibition on the sympatho-vagal contol of heart rate. J Auton Nerv Syst (1998) 73:63–73.[CrossRef][ISI][Medline]
  33. Sobey C.G., Faraci F.M. Effect of nitric oxide and potassium channel agonists and inhibitors on basilar artery diameter. Am J Physiol (1997) 272:H256–H262.[ISI][Medline]
  34. Takata Y., Shimada F., Kato H. Differential effects of diazoxide, cromakalim and pinacidil on adrenergic neurotransmission and 86Rb+ efflux in rat brain cortical slices. J Pharmacol Exp Ther (1992) 263:1293–1301.[Abstract/Free Full Text]
  35. Takata Y., Shimada F., Kato H. Possible involvement of ATP-sensitive K+ channels in the inhibition of rat central adrenergic neurotransmission under hypoxia. Jpn J Pharmacol (1993) 62:279–287.[Medline]
  36. Tomai F., Crea F., Chiariello L., Gioffre P.A. Ischemic preconditioning in humans: models, mediators, and clinical relevance. Circulation (1999) 100:559–563.[Abstract/Free Full Text]
  37. Vanheel B., Van de Voorde J. Nitric oxide induced membrane hyperpolarization in the rat aorta is not mediated by glibenclamide-sensitive potassium channels. Can J Physiol Pharmacol (1997) 75:1387–1392.[CrossRef][ISI][Medline]
  38. von der Weid P.Y. ATP-sensitive K+ channels in smooth muscle cells of guinea-pig mesenteric lymphatics: role in nitric oxide and beta-adrenoceptor agonist-induced hyperpolarizations. Br J Pharmacol (1998) 125:17–22.[CrossRef][ISI][Medline]
  39. Weiss J.N., Venkatesh N., Lamp S.T. ATP-sensitive K+ channels and cellular K+ loss in hypoxic and ischaemic mammalian ventricle. J Physiol (Lond) (1992) 447:649–673.[Abstract/Free Full Text]

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



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
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
Google Scholar
Right arrow Articles by Mohan, R. M
Right arrow Articles by Paterson, D. J
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mohan, R. M
Right arrow Articles by Paterson, D. J
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?