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Cardiovascular Research 2005 67(1):60-68; doi:10.1016/j.cardiores.2005.03.011
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Copyright © 2005, European Society of Cardiology

Kir6.2-deficient mice are susceptible to stimulated ANP secretion: KATP channel acts as a negative feedback mechanism?

Noriko Saegusaa,b, Toshiaki Satoa, Tomoaki Saitoa, Masaji Tamagawaa, Issei Komurob and Haruaki Nakayaa,*

aDepartment of Pharmacology, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan
bDepartment of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan

* Corresponding author. Tel.: +81 43 226 2051; fax: +81 43 226 2052. Email address: nakaya{at}faculty.chiba-u.jp

Received 20 December 2004; revised 16 February 2005; accepted 11 March 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: While atrial natriuretic peptide (ANP) has been shown to be released mainly from cardiac muscle cells in response to atrial distension, the regulatory mechanisms of ANP secretion are still not fully understood. We sought to determine whether the ATP-sensitive K+ (KATP) channel modulates the secretion of ANP, using mice with homozygous knockout of the Kir6.2 (a pore-forming subunit of cardiac KATP channel) gene.

Methods: KATP channel currents were recorded from isolated mouse atrial cells with patch–clamp techniques. Plasma ANP concentrations in anesthetized mice and ANP content and secretion in isolated atrial preparations were determined by radioimmunoassay. Action potentials were recorded from the isolated atria.

Results: Exposure to 2,4-dinitrophenol (100 µM) evoked a glibenclamide-sensitive KATP channel current in atrial cells from wild-type (WT) but not Kir6.2 knockout (Kir6.2 KO) mice. Although there were no significant differences in the basal plasma ANP levels between WT and Kir6.2 KO mice, volume expansion caused a significant elevation of plasma ANP concentration in Kir6.2 KO but not WT mice with accompanying hypotension. When isolated left atria were stretched, ANP secreted into the bath from Kir6.2 KO atria was significantly higher than that from WT atria. Furthermore, stretching the atria from WT but not Kir6.2 KO mice significantly shortened the action potential duration. A hypotonic stretch of the membrane induced the glibenclamide-sensitive KATP channel current in atrial cells from WT but not Kir6.2 KO mice.

Conclusions: Kir6.2 is essential for the function of KATP channel in mouse atrial cells. Given that Kir6.2 KO mice are susceptible to stretch-induced secretion of ANP, our results suggest that KATP channels may act as a negative feedback mechanism for the control of ANP secretion.

KEYWORDS Natriuretic peptide; K-ATP channel; Myocytes

This article is referred to in the Editorial by L.G. Bianciotti (pp. 9–10) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
ATP-sensitive K+ (KATP) channels, originally discovered in cardiac muscle [1], are present in many tissues and play an important role in various cellular responses [2]. The molecular identity of sarcolemmal KATP channels is now known to be a hetero-octameric complex of four pore-forming subunits (Kir6.x) and four sulfonylurea receptor regulatory subunits (SUR) [2]. Previous studies have shown that mutant mice lacking the Kir6.2 subunit of KATP channels (Kir6.2 KO mice) display obvious impairment of insulin response to glucose [3] and are susceptible to generalized seizures after brief hypoxia [4]. Our recent studies using the Kir6.2 KO mice have provided direct evidence that Kir6.2 forms the pore region of ventricular KATP channels [5] and the activation of KATP channels plays an important role in cardioprotection [6]. More recently, Kir6.2 KO mouse models highlight the importance of KATP channels in adaptation to stress beyond their role in cytoprotection [7].

Although considerable advances have been made in recent years towards understanding the nature of ventricular KATP channels, the molecular identity and functional role of atrial KATP channels are poorly understood. Atrial distention causes release into the circulation of atrial natriuretic peptide (ANP), a hormone that plays a role in the regulation of cardiovascular homeostasis [8]. Since the gating of the atrial KATP channel is mechanosensitive [9], the relationship to the process of ANP secretion is a subject of considerable interest. Kim et al. [10] reported that the KATP channel blocker glibenclamide suppressed the stretch-stimulated ANP secretion from rat atria. On the other hand, conflicting observation was reported that the KATP channel blocker tolbutamide increased the release of ANP in neonatal rat atrial myocytes [11]. Moreover, Xu et al. [12] reported that the stretch-induced ANP secretion was inhibited by KATP channel openers. Thus, the results so far obtained by using KATP channel blockers and openers are not conclusive. In this study, we sought to determine whether atrial KATP channel regulates the secretion of ANP, using Kir6.2-deficient mice. The results show that Kir6.2 is essential for the function of atrial KATP channel and Kir6.2-deficient mice are susceptible to stretch-induced secretion of ANP.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Kir6.2-deficient mice
All procedures were performed in conformity with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, revised 1996) and were approved by the Institutional Animal Care and Use Committee of Chiba University. A mouse line deficient in the KATP channels was generated by targeted disruption of the gene coding for Kir6.2, as described previously [3]. C57BL/6 mice were used as control because they had been back-crossed to a C57BL/6 strain for five generations. Twelve- to fourteen-week-old Kir6.2-deficient mice or C57BL/6 control mice were used in this study.

2.2. Electrophysiology
Single atrial cells were enzymatically isolated by the method of Suzuki et al. [5] with some modifications. Single-channel and whole-cell membrane currents were recorded by the patch–clamp method as previously described [5,6]. Whole-cell current recordings were performed at 36 °C with nystatin in the pipette solution. Single atrial cells were superfused with HEPES–Tyrode's solution (in mM): NaCl 143, KCl 5.4, CaCl2 1.8, MgCl2 0.5, NaH2PO4 0.33, glucose 5.5, and HEPES–NaOH buffer 10 (pH=7.4). The pipette solution contained (in mM): K-aspartate 110, KCl 20, MgCl21.0, CaCl2 1.0, EGTA 0.1, and HEPES–KOH buffer 5 (pH=7.4), with 250 µg/ml nystatin. A ramp-pulse protocol (–100 to +50 mV over 2.5 s, repeated at 30-s intervals) was used to record the quasi-steady-state membrane current. For the experiments of hypotonic stretch of the membrane (Fig. 6), the isolated atrial cells were exposed to the low-chloride isotonic or hypotonic solution in order to minimize swelling-induced chloride current contamination [13]. Isotonic solution contained (in mM): Na-aspartate 80, KCl 5.4, CaCl2 1.8, MgCl2 0.5, NaH2PO4 0.33, glucose 5.5, mannitol 119, and HEPES–NaOH buffer 10 (pH=7.4, {approx}36 °C) with an osmolarity of 300 mosM/kg H2O. Hypotonic solution was prepared by simply omitting mannitol from the isotonic solution with the osmolality of 180 mosM/kg H2O. For single-channel recordings from the cell-attached patches, symmetrical high K+ external and internal solutions ({approx}22 °C) were used. The external solution contained (in mM): KCl 140, EGTA 5, Na2–ATP 0.1, and HEPES–KOH buffer 5 (pH=7.4) and the pipette solution contained (in mM): KCl 140, MgCl2 1.8, CaCl2 1.8, and HEPES–KOH buffer 5 (pH=7.4). The current signals were digitized at 2 kHz for data analysis with pClamp software (Axon Instruments, Foster City, CA).


Figure 6
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  		Fig. 6 Activation of the atrial KATP current by hypotonic stretch. (A) Original whole-cell voltage-ramp currents traces of atrial cells from WT (left) and Kir6.2 KO mice (right). (B) Glibenclamide-sensitive current densities at 0 mV in WT (n=4) and Kir6.2 KO (n=4) atrial cells are summarized. Values are mean ± SEM. *p<0.05 vs. control.

 
2.3. Volume expansion
The mice were anesthetized with urethane (1.5 g/kg, i.p.) and placed on a heating pad to maintain rectal temperatures at 37 °C. The left external jugular vein was cannulated with small polyethylene catheters for intravenous infusion. Volume expansion was performed by a modification of the method of Kishimoto et al. [14]. The lactated Ringer's solution containing 4.5% bovine serum albumin was infused at a rate of 200 µl/h/g (body weight) for 30 min. The rate of infusion was then lowered to 4.3 µl/h/g for another 30 min. In some experiments the initial infusion rate was increased from 200 to 400 µl/h/g for 30 min, after which the infusion was continued at a rate of 4.3 µl/h/g for 30 min. These infusions were carried out with a microinfusion pump (Harvard Apparatus, MA). At the end of the volume expansion, blood (700 µl) was drawn from the right carotid artery, collected into a tube containing EDTA and aprotinin, and centrifuged for 20 min at 4 °C. Plasma samples were stored at –80 °C until analysis by ANP radioimmunoassay.

2.4. Hemodynamic measurements
Arterial blood pressure was measured continuously in mice anesthetized with urethane. The right carotid artery was cannulated with small polyethylene catheters for the measurement of blood pressure via a pressure transducer. Heart rate was derived from the arterial blood pressure signal.

2.5. Isolated atrial preparations
Tissue bath preparations of left atria were prepared by a modification of the method described by Bilder et al. [15]. Briefly, left atria from WT and Kir6.2 KO mice were dissected free and placed in a 3-ml water jacketed tissue bath containing HEPES–Tyrode's solution (pH=7.4) gassed with 100% O2 (37 °C). The edge of preparation was pinned to the rubber base of the tissue bath and electrically paced at a frequency of 5 Hz. After 10 min equilibration, the bath solution was discarded and 1 ml of fresh HEPES–Tyrode's solution was added to the bath. Atrial preparations were incubated in this solution with or without stretch stimulus. The atria connected to a string were stretched with a fixed resting tension of 0.5 g. After 10 min of incubation, the bath solution was collected into a tube containing EDTA and aprotinin and was stored at –80 °C until analysis by ANP radioimmunoassay.

2.6. ANP content of the atria
The hearts were rapidly removed from the anesthetized mice and the left atria were dissected out and immediately frozen in liquid nitrogen. The lysates were obtained from three preparations by homogenization in ice-cold buffer (0.1 mM acetic acid). The lysates were incubated for 8 min at 100 °C, kept on ice for 15 min and cleared by centrifugation at 66,000 x g for 20 min at 4 °C. Protein concentration was determined by the BCA protein assay kit (PIERCE, Rockford, IL).

2.7. ANP radioimmunoassay
The concentration of immunoreactive ANP was measured by commercially available radioimmunoassay, as described previously [16]. The threshold for detection of ANP was 1.5 pg/ml.

2.8. Action potential recordings
The preparations of left atria from WT and Kir6.2 KO mice were mounted in a recording chamber and perfused at a constant flow (5 ml/min) with Tyrode's solution (in mM): NaCl 125, KCl 4.0, CaCl2 2.7, MgCl2 0.5, NaH2PO4 1.8, glucose 5.5, and NaHCO3 25 and gassed with 95% O2/5% CO2 (37 °C). Action potentials were evoked by electrical field stimulation at 5 Hz (2-ms rectangular pulses at 2 x threshold intensity) and recorded by use of a 3 M KCl-filled microelectrode (tip resistance 10–20 M{Omega}). The edge of left atrial preparation was stretched with a fixed resting tension of 0.5 g. Transmembrane potential was recorded by a direct current preamplifier (MEZ-7200, Nihon Kohden, Tokyo, Japan) and digitized (PowerLab 2/20, ADInstruments, Castle Hill, Australia).

2.9. Drugs
The following drugs were used: nystatin (Wako Pure Chemicals, Osaka, Japan) and glibenclamide (Sigma-Aldrich Japan, Tokyo, Japan). Nystatin was dissolved in methanol at a concentration of 10 mg/ml and added to the pipette solution at a concentration of 250 µg/ml just before experiments. Glibenclamide was dissolved in dimethyl sulfoxide as a stock solution of 10 mM and final concentration of dimethyl sulfoxide was less than 0.1%.

2.10. Statistics
All data are presented as mean ± SEM. Statistical comparisons were made with the use of Student's t test or ANOVA combined with Fisher post hoc test, as appropriate. A p value of less than 0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. KATP channel function in atrial cells
We first evaluated KATP channel function in atrial cells isolated from wild-type (WT) and Kir6.2-deficient (Kir6.2 KO) mice. Whole-cell membrane currents were recorded using a ramp-pulse protocol (Fig. 1A and B). There were no significant differences in the density of the outward current at 0 mV between atrial cells isolated from WT (1.4 ± 0.1 pA/pF, n=6) and Kir6.2 KO mice (1.6 ± 0.1 pA/pF, n=6) in the control condition. In WT cells, metabolic inhibition with a glucose-free, 100 µM DNP-containing solution induced an outward current (9.1 ± 0.1 pA/pF at 0 mV, n=6), which, by virtue of its blockade by 1 µM glibenclamide (1.3 ± 0.1 pA/pF, n=6), was confirmed to be the KATP current. However, such a membrane current change was not observed in Kir6.2 KO cells (1.6 ± 0.1 pA/pF after metabolic inhibition, n=6). When the atrial cells were exposed to a solution containing DNP (100 µM), single KATP channel activity could be recorded from 25 of 25 cell-attached patches of 9 WT mice (Fig. 1C). The channel openings were inhibited by addition of 1 µM glibenclamide to the solution (data not shown). The linear slope conductance, obtained from the current–voltage relationship from –100 to –40 mV for the single channel current of WT cells, was 71.6 ± 1.7 pS (n=7). In contrast, opening of KATP channels could not be recorded from any cell-attached patches of Kir6.2 KO cells (n=18). These results indicate that Kir6.2 is essential for the function of KATP channel in atrial cells.


Figure 1
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  		Fig. 1 (A) Effect of metabolic inhibition with a glucose-free, DNP-containing (100 µM) solution and co-application of glibenclamide (GLB, 1 µM) on the nystatin-perforated whole-cell membrane currents recorded from atrial cells of WT (left) and Kir6.2 KO mice (right). (B) Current densities at 0 mV from control (n=6 of WT, n=6 of Kir6.2 KO, black bar), DNP-treated (n=6 of WT, n=6 of Kir6.2 KO, grey bar) and DNP+GLB (n=6 of WT, dark grey bar) mice of atrial cells are summarized. Values are mean ± SEM. *p<0.01 vs. control. (C) Single-channel current recordings in the cell-attached mode by addition of DNP (100 µM) from atrial cells of WT (left) and Kir6.2 KO mice (right). The test potential is indicated on the left.

 
3.2. Effect of volume expansion on plasma ANP level
In anesthetized mice, there were no significant differences in the basal plasma ANP concentrations between WT (28.7 ± 3.0 pg/ml, n=7) and Kir6.2 KO mice (36.6 ± 4.9 pg/ml, n=6). After volume expansion (200 µl/h/g for 30 min and 4.3 µl/h/g for another 30 min), the plasma ANP level was increased to 31.8 ± 1.6 pg/ml (n=7) in WT mice, but this change was not statistically significant (p=0.385 vs. baseline). On the other hand, Kir6.2 KO mice reacted to volume expansion and the plasma ANP concentration significantly increased to 73.5 ± 13.4 pg/ml (n=5, p=0.021 vs. baseline). The elevation of plasma ANP levels observed in Kir6.2 KO mice was notably higher than that observed in WT mice (p=0.004, Fig. 2). When the initial infusion rate was increased from 200 to 400 µl/h/g for 30 min (then the rate of infusion was lowered to 4.3 µl/h/g for 30 min) to give a severe volume expansion, the plasma ANP concentration in WT mice significantly increased to 49.8 ± 8.2 pg/ml (n=4, p=0.024 vs. baseline). This value was not significantly different from that in Kir6.2 KO mice (53.9 ± 4.9 pg/ml, n=4). These results indicate that Kir6.2 KO mice are susceptible to the ANP secretion by volume expansion.


Figure 2
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  		Fig. 2 Effect of volume expansion on plasma ANP levels of WT and Kir6.2 KO mice. Top panel shows the experimental protocol. Bottom panel shows summarized data for plasma ANP concentration. Blood samples were drawn at baseline (n=7 of WT, n=6 of Kir6.2 KO, grey bar) and after volume expansion (n=7 of WT, n=5 of Kir6.2 KO, black bar). Values are mean ± SEM.

 
3.3. Hemodynamic effects of volume expansion
There were no significant differences in the basal values of mean arterial pressure (MAP) between WT and Kir6.2 KO mice (Fig. 3). The values of MAP at 30 min of volume expansion were similarly increased in both WT (112.0 ± 4.1% of baseline, n=4) and Kir6.2 KO mice (111.3 ± 2.3% of baseline, n=4). In WT mice, MAP returned to baseline levels after 60 min of volume expansion (96.7 ± 6.2% of baseline, n=4). In contrast, 60 min of volume expansion in the Kir6.2 KO mice produced significant decrease in MAP (81.0 ± 4.7% of baseline, n=4, p<0.05). There were no significant differences in the basal values of heart rate between WT (654 ± 31 bpm, n=7) and Kir6.2 KO mice (642 ± 50 bpm, n=5). Volume expansion did not alter the heart rate in both WT (689 ± 25 bpm, n=7) and Kir6.2 KO mice (644 ± 50 bpm, n=5).


Figure 3
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  		Fig. 3 Effect of volume expansion on mean arterial pressure (MAP) in anesthetized mice. (A) Changes of MAP before (baseline) and after 30 and 60 min of volume expansion. (B) MAPs are given as a percentage of the value measured at baseline. Data represent mean ± SEM of 4 experiments for each group. *p<0.05 vs. baseline, #p<0.05 vs. 30 min.

 
3.4. ANP secretion from isolated atria
Isolated mouse atrial preparations were used to study the direct effect of mechanical stretch on ANP release. As shown in Fig. 4, in most cases the ANP concentration did not exceed the threshold for detection (≥ 1.5 pg/ml). We therefore repeated the experiment until the ANP secretion was detected in 4 or 5 of the preparations in each group. In control (non-stretch) conditions, ANP secretion was detected in 4 of 17 atria from WT and in 5 of 13 atria from Kir6.2 KO, respectively. The mean ANP concentration in each of 4 or 5 preparations (WT: 2.2 ± 0.2 pg/ml, Kir6.2 KO: 2.8 ± 0.4 pg/ml) was not statistically significant. After mechanical stretch for 10 min, ANP secretion was detected in 4 of 15 atria from WT. In Kir6.2 KO atria, ANP secretion was detected in 4 of 8 preparations and the mean ANP concentration (4.9 ± 0.7 pg/ml) was notably higher that that observed in WT atria (2.4 ± 0.3 pg/ml, p=0.046).


Figure 4
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  		Fig. 4 Effects of mechanical stretch on ANP secretion in isolated atria from WT and Kir6.2 KO mice. The threshold for detection of ANP (1.5 pg/ml) is represented as dotted line. Each symbol represents data from individual preparations. Closed squares indicate the mean and the vertical bars indicate SEM.

 
ANP content of atria obtained from WT and Kir6.2 KO mice was 0.64 ± 0.30 ng/mg protein and 0.74 ± 0.23 ng/mg protein, respectively; these values did not reach statistical significance (n=3, p=0.696).

3.5. Action potentials in isolated atria
Representative recordings of action potentials before and after 5 min of stretch are shown in Fig. 5A and B. The action potential duration (APD) in the WT atrium was shortened at 5 min of stretch, while the APD remained unaltered in the Kir6.2 KO atrium. As summarized in Fig. 5C, stretching the WT atria significantly shortened the APD measured at 90% repolarization (APD90) to 71.8 ± 3.4% of control (n=4, p=0.004). Stretch-induced shortening of APD90 was not observed in Kir6.2 KO atria (98.6 ± 0.7%, n=4).


Figure 5
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  		Fig. 5 Representative recording of action potentials before (Control, black) and 5 min after stretch (Stretch, grey) in the atrium of WT (A) and Kir6.2 KO (B) mice. (C) Summarized effects of stretch on the action potential duration. The action potential duration at 90% repolarization (APD90) was measured 5 min after stretch and given as a percentage of the control value. Data represent mean ± SEM of 4 experiments for each group. *p<0.05 vs. control.

 
3.6. Hypotonic stretch-induced KATP current
To determine whether KATP channel currents could be mechanically induced, the nystatin-perforated patch was used to record whole-cell membrane currents and atrial cells were perfused with either isotonic or hypotonic solutions. Fig. 6A shows the representative current traces recorded in response to voltage ramps from –100 to +50 mV. In an atrial cell of WT, a hypotonic stretch of the membrane for 10 min evoked an outward current that could be reduced by the subsequent application of glibenclamide (1 µM). The reversal potential of the glibenclamide-sensitive current, which isolated by digital subtraction of the current trace in the presence of glibenclamide from that under hypotonic condition, was close to the K+ equilibrium potential (~–80 mV, data not shown). On the other hand, a hypotonic stretch of the membrane slightly increased the outward current in Kir6.2 KO cells, but this current was not blocked by glibenclamide. As summarized in Fig. 6B, hypotonic stretch of WT cells significantly increased the glibenclamide-sensitive outward current at 0 mV from 0.23 ± 0.03 to 1.73 ± 0.36 pA/pF (n=4, p<0.05). However, in Kir6.2 KO cells, a glibenclamide-sensitive outward current was not evoked under hypotonic stretch (from 0.13 ± 0.03 to 0.16 ± 0.07 pA/pF, n=4, p=0.66). These results indicate that KATP channel current is activated by hypotonic stretch of the membrane in atrial cells from WT but not Kir6.2 KO mice.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
4.1. Kir6.2 forms the pore of atrial KATP channels
A previous study using primary cultured neonatal rat atrial cells suggested that molecular composition of atrial KATP channels may be different from that of ventricular KATP channels based on the functional and pharmacological profiles [17]. In the present study, however, a glibenclamide-sensitive KATP channel current could be observed during metabolic inhibition in atrial cells of WT but not Kir6.2 KO mice (Fig. 1A and B). In addition, we found that the unitary conductance of single KATP channel current in WT mouse atrial cells (71.6 ± 1.7 pS, n=7, Fig. 1C) was close to that of ventricular cells (75.9 ± 1.3 pS, n=7, data not shown). Therefore, it can be concluded that Kir6.2 is essential for the function of mouse atrial KATP channels and Kir6.2 KO mice are potentially useful to examine whether KATP channel modulates the secretion of ANP.

4.2. KATP channel modulates ANP secretion in vivo and in vitro
It is acknowledged that ANP is stored as pro-ANP within the granules of cardiomyocytes and released in response to atrial stretch [18,19]. Pro-ANP is cleaved during the release process by a cardiac protease, corin, to form the biologically active C-terminal ANP [20]. Here we examined, in vivo and in vitro, whether KATP channel is crucial for the regulation of ANP secretion. Plasma volume expansion was used as a means of producing release of the cardiac ANP granules in vivo. There were no significant differences in the basal plasma ANP levels (Fig. 2) between WT and Kir6.2 KO mice. After the initial volume expansion (200 µl/h/g for 30 min), MAP in WT and Kir6.2 KO mice increased to similar extents (Fig. 3), suggesting that the volume expansion seemed to be equally effective in both WT and Kir6.2 KO mice. In WT mice, however, volume expansion was insufficient to cause a significant increase in plasma ANP concentration. Despite a rather mild volume expansion, a significant increase of plasma ANP concentration was observed in Kir6.2 KO mice (Fig. 2). ANP promotes diuresis/natriuresis and reduces vascular tone [8]. Consequently, hypotension might occur in Kir6.2 KO but not in WT mice (Fig. 3), accompanied by an excessive secretion of ANP.

ANP secreted into the bath from atrial tissue in vitro was small and ≥ 30% of preparations exceeded the threshold for detection in control conditions. However, 4 of 8 preparations (50%) from Kir6.2 KO mice exceeded the threshold for detection after mechanical stretch and the ANP concentration was greater than that of WT atria (Fig. 4). Thus, in vitro study using isolated atria was consistent with the findings in vivo. Since ANP content of WT atria was not statistically different from Kir6.2 KO atria, in vivo and in vitro studies suggest that Kir6.2 KO mice are susceptible to stretch-induced secretion of ANP. Furthermore, given the fact that an excess of ANP produced hypotension in Kir6.2 KO mice, it is reasonable to suppose that activation of KATP channels is a mechanism for feedback inhibition of stimulated ANP release.

4.3. Activation of atrial KATP channels by mechanical stretch
Based on pharmacological experiments, it has been proposed that KATP channels regulate ANP secretion [10,11]. In the present study, we found that mechanical stretch shortened the action potential duration in WT atria, but not in Kir6.2 KO atria (Fig. 5). Van Wagoner [9] reported that KATP channels in rat atrial cells were mechanosensitive and activated by a hypotonic swelling. Later on, Baron et al. [17] also demonstrated a hypotonic-induced activation of the atrial appendage KATP channel currents. In agreement with previous reports, we could record the glibenclamide-sensitive KATP channel current in response to a hypotonic stretch in atrial cells of WT but not of Kir6.2 KO mice (Fig. 6). Although we did not investigate the hypotonic stretch-induced ANP secretion, Jiao et al. [11] have demonstrated that a hypotonic swelling increases ANP secretion in rat atrial myocyte culture. Together, these findings suggest that mechanical stimulus of atrial myocytes activates KATP channel in association with ANP secretion.

4.4. Possible mechanisms underlying KATP channel-mediated regulation of ANP secretion
Our observations raise the question of how stretch-induced opening of KATP channels prevents excessive release of ANP. The diagram of Fig. 7 shows the possible mechanism underlying KATP channel-mediated regulation of ANP secretion. Mechanical stretch in isolated atrial tissues was reported to increase the intracellular Ca2+ transients and the late duration of the action potentials, which was ascribed to the stretch-induced channel activation and resultant augmentation of the Na+/Ca2+-exchanger inward current [21,22]. It has also been proposed that intracellular Ca2+ plays a critical role in ANP secretion from atrial cells and changes of cytosolic Ca2+ concentration affect ANP secretion [8,23,24]. Activation of atrial KATP channels by mechanical stretch may decrease the cytosolic Ca2+ concentration by abbreviating the action potentials, which is expected to reduce the time for Ca2+ influx via L-type Ca2+ channels and to increase the time for Ca2+ extrusion through the Na+/Ca2+-exchange system. Whatever the mechanism involved, the present study clearly demonstrates that KATP channels negatively regulate ANP secretion from atrial tissues.


Figure 7
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  		Fig. 7 Hypothetical representation of mechanism by which opening of KATP channels acts as a negative modulator of ANP secretion. Mechanical stretch in atrial tissue increases the intracellular Ca2+ concentration ([Ca2+]i) and thereby stimulates the release of ANP. On the other hand, KATP channel opens when stretched and decreases [Ca2+]i by shortening of action potential duration (APD), which in turn inhibits the release of ANP. (+): stimulation, (–): inhibition. (See text for details.)

 
4.5. Conclusions
ANP is known to produce a variety of physiological actions such as diuretic, vasodilative [25,26], anti-ischemic [27] and antihypertrophic actions [28]. In the present study we have provided evidence that Kir6.2 is essential for the function of KATP channel in atrial myocytes and that KATP channel-dependent mechanism contributes to the regulation of ANP secretion. Recently, a functional study using Kir6.2 KO mice has demonstrated that disruption of KATP channel function leads to impaired Ca2+ handling, cardiac dysfunction and lethal arrhythmias under vigorous sympathetic stimulation, suggesting requirement of KATP channels for adaptation to physiological stress [7]. Furthermore, it has been reported that in failing hearts the metabolic dysregulation of KATP channels can occur and resultant loss of protective mechanisms expands the risk of disease progression [29]. Therefore, ANP secretion induced by impaired KATP channel function may play a compensatory role by protecting the heart under pathophysiological states. It is likely, however, that under physiological conditions KATP channel may act as a negative feedback mechanism for the control of ANP secretion.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This study was supported in part by Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science, the Mitsui Life Social Welfare Foundation, K. Watanabe Research Foundation, and the Vehicle Racing Commemorative Foundation. We are grateful to Drs. S. Seino and T. Miki, Kobe University, for generous donation of Kir6.2 KO mice. We also thank Dr. H. Uemura and Dr. T. Ogura for helpful discussion and Ms. Y. Reien and I. Sakashita for excellent technical and secretarial assistance.


   Notes
 
Time for primary review 21 days


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

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