This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tsuchiya, K. Articles by Sasayama, S. Search for Related Content PubMed PubMed Citation Articles by Tsuchiya, K. Articles by Sasayama, S.

(Circulation. 1997;96:3129-3135.)
© 1997 American Heart Association, Inc.

Articles

Functional Compartmentalization of ATP Is Involved in Angiotensin II–Mediated Closure of Cardiac ATP-Sensitive K⁺ Channels

Kunihiko Tsuchiya, MD; Minoru Horie, MD, PhD; Masato Watanuki, MD; Carlos A. Albrecht, MD; Kazuhiko Obayashi, MD; Hisayoshi Fujiwara, MD, PhD; ; Shigetake Sasayama, MD, PhD

From the Department of Cardiovascular Medicine (K.T., M.H., M.W., C.A.A., K.O., S.S.), Kyoto University Graduate School of Medicine, Kyoto 606-01; and the Second Department of Internal Medicine (H.F.), Faculty of Medicine, Gifu University, Gifu 500, Japan.

Correspondence to Minoru Horie, MD, PhD, Division of Cardiac Electrophysiology, The Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, Shogoin, Sakyo-ku, Kyoto 606-01, Japan. E-mail horie{at}kuhp.kyoto-u.ac.jp

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background The effects of angiotensin II (Ang II) on ATP-sensitive K⁺ channels (K_ATP) were investigated in ventricular myocytes enzymatically isolated from adult guinea pig heart.

Methods and Results In the whole-cell and cell-attached configurations (including open-cell-attached mode) of the patch-clamp technique, K_ATP currents (I_KATP) were activated through metabolic poisoning by the use of inhibitors of both glycolytic and oxidative ATP productions at 37°C. In the whole-cell mode, I_KATP were reversibly suppressed by increasing extracellular glucose and Ang II (1 nmol/L). In the cell-attached mode, Ang II concentration-dependently inhibited single K_ATP activities with an IC₅₀ value of 3.2±0.5 pmol/L (Hill coefficient=1.3±0.3). CV11974 (100 nmol/L), an angiotensin 1 (AT₁) receptor-selective antagonist, blocked the inhibitory action of Ang II. Preincubation of myocytes with pertussis toxin (5 µg/mL for >120 min at 37°C) virtually prevented subsequent Ang II action. The inhibitory effect of Ang II was also abolished in the open-cell–attached mode (achieved by a prior perfusion of streptolysin-O, 0.08 U/mL). In this mode, through tiny membrane holes, the intracellular ATP concentration can be controlled by bathing extracellular solutions containing a known ATP concentration.

Conclusions The inhibitory actions of Ang II on K_ATP appear to be mediated by an increase in the subsarcolemmal ATP concentration that results from the inhibition of adenylate cyclase activities via AT₁ receptors/PTX-sensitive G proteins.

Key Words: angiotensin • ischemia • potassium channels

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Ang II is an octapeptide hormone and principal effector of the RAS.^{1 2 3 4 5} The concept of RAS has recently been expanded since local or tissue RAS was identified in a variety of organs, including the heart: (1) angiotensin I is converted to Ang II in isolated, perfused rat heart^{6 7} ; (2) Ang II receptors are found in the hearts of several animals, including humans^{8 9 10 11} ; and (3) genes encoding renin and angiotensinogen are coexpressed in myocytes.¹² The clinical significance of cardiac RAS has been revealed through the effects of ACE inhibitors.^{13 14}

The secretory level of Ang II is increased in pathological conditions such as coronary vasospasm and myocardial ischemia and infarction⁶ and may be associated with modulated cardiac function under these conditions. Ang II receptors couple to the major phosphorylation systems—negatively to protein kinase A^{15 16} and positively to protein kinase C.^{17 18} However, direct linkage between RAS and cardiac electrical properties has not been fully elucidated.

Under metabolic stress, K_ATP¹⁹ activate and serve as metaboelectrical sensors, thereby regulating the duration of action potential, Ca²⁺ influx, and myocardial contractility.^{20 21} The activation of the channels minimizes the infarct size in several animal models.^{22 23}

We therefore examined whether Ang II stimulation modulates K_ATP activity in guinea pig ventricular myocytes. We found that Ang II reversibly inhibits K_ATP through an increase in the subsarcolemmal ATP concentrations ([ATP]), which results from the inhibition of adenylate cyclase activity through PTX-sensitive G proteins coupled to AT₁ receptors.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Preparation of Single Ventricular Myocytes
Adult guinea pigs of either sex (250 to 400 g) were anesthetized with an injection of pentobarbital sodium (40 to 50 mg/kg IP). Ventricular myocytes were isolated according to the collagenase perfusion method as previously described.²⁴ Briefly, the chest was opened under artificial ventilation, and the aorta was cannulated. The heart was then quickly dissected. Using a Langendorff apparatus, the heart was perfused with normal Tyrode's solution for 5 minutes to wash out the blood, followed by nominally Ca²⁺-free Tyrode's solution for 3 minutes to stop the heartbeat. The heart was then switched to nominally Ca²⁺-free Tyrode's solution containing 0.4 mg/mL collagenase (Type I, Sigma Chemical) for 20 minutes and rinsed with a high-K⁺, low-Ca²⁺ "KB solution"²⁴ for 3 minutes. The composition of Tyrode's solution was (in mmol/L) 143 NaCl, 0.3 NaH₂PO₄, 5.4 KCl, 0.5 MgCl₂, 1.8 CaCl₂, and 5 HEPES/NaOH (pH 7.4 adjusted with NaOH), and the composition of KB solution (in mmol/L) was 70 L-glutamic acid, 25 KCl, 20 taurine, 10 KH₂PO₄, 3 MgCl₂, 0.5 EGTA, 11 glucose, and 10 HEPES (pH 7.3 with KOH).

The left ventricle was gently dissected into small pieces in KB solution at room temperature. The cell suspension was passed through a 105-µm mesh filter, and single cells obtained after centrifugation at 400 rpm for 3 minutes were preincubated at 37°C for >120 minutes in KB solution containing the metabolic inhibitor 2-deoxyglucose (5.5 mmol/L; Nacalai Tesque) instead of glucose to suppress ATP production by glycolysis. For the experiments with metabolic stress, the myocytes were preincubated with 2-deoxyglucose for >60 minutes at 36°C (5.5 mmol/L).

Electrophysiology
Patch pipettes were prepared by pulling borosilicate glass capillaries (Hilgenberg) at a resistance of 2 to 3 M for whole-cell mode and 4 to 5 M for single-channel mode experiments. In the whole-cell mode, the pipette solution contained (in mmol/L) 130 aspartic acid, 10 KCl, 10 NaCl, 1 MgCl₂, 10 EGTA, and 0.1 K₂ATP (pH 7.4 adjusted with 5 HEPES/KOH). I_KATP were activated by perfusing glucose- and Ca²⁺-free Tyrode's solution containing 1 mmol/L NaCN, monitored by applying ramp pulses (±100 mV, -100 mV/s) from a holding potential of -40 mV. The current data obtained with a patch-clamp amplifier (AXOPATCH 200A, Axon Instruments) were directly displayed on a chart recorder (Linearcorder WR 3320; Graphtec) with on-line acquisition to an NEC personal computer at a Bessel-type cutoff frequency of 0.5 kHz at a sample frequency of 1 kHz.

In the cell-attached, open-cell–attached, and inside-out conditions, normal Tyrode's medium without glucose or Ca²⁺ was used as pipette solution, and glucose-free, K⁺-rich solution containing 1 mmol/L NaCN (except inside-out ) and 0.5 mmol/L EGTA was used as external solution to block the glycolytic ATP production and minimize the influence of PKC-dependent pathway. Single-channel activities were re-corded by a patch-clamp amplifier (AXOPATCH 200A) with a simultaneous back-up onto a videotape via a pulse-coded modulation converter system (NF RP880) for later off-line analysis. K_ATP were activated by superfusing myocytes with external solution. The composition was (in mmol/L) 150 KCl, 0.5 EGTA, and 5 HEPES/KOH (pH 7.4 adjusted with KOH). The transmembrane potential was virtually eliminated in this solution.

Drugs
Ang II (Peptide Institute), CV11974 (Takeda Chemical Industry), PD123319 (Parke-Davis), ouabain, and isoproterenol (Nacalai Tesque) were prepared as stock solutions in distilled water and further diluted with test solutions immediately before use. PTX (Seikagaku Co) was dissolved in a KB solution (50 µg/mL stock) and diluted for the myocyte suspension at a final concentration of 5 µg/mL. Incubation was carried out at 37°C for >120 minutes.

Stock solutions of glibenclamide (1 mmol/L; Hoechst) and cromakalim (100 mmol/L, Taisho Pharmaceutical Co) were prepared in dimethylsulfoxide. Dimethylsulfoxide alone (<0.1%) had no effects on membrane currents. Streptolysin-O (Wellcome) was prepared at a concentration of 0.08 U/mL for the open-cell–attached mode.²⁵ All other reagents were purchased from Sigma Chemical Co or Wako Industries Ltd. All experiments were performed at 35° to 37°C.

Data Analysis
The mean patch current (I) for single-channel events was measured as the average difference between baseline currents (all channels closed) and open channel currents. The unitary amplitude of open channel currents (i) was estimated by analysis of the amplitude distribution using an NEC personal computer at a Bessel-type cutoff frequency of 0.5 kHz at a sample frequency of 1 kHz.

The mean number of open channels (NP) was given by the product of the number of available channels in the patch (N) and the probability of their being open (P). NP was estimated by dividing the mean patch currents (I=NPi) by unitary current (i) as I/i. The NP values were measured in the presence of various concentrations of Ang II and normalized by the NP obtained in the absence of Ang II (NP_o). The calculated relative channel activities (%NP/NP_o) were plotted against Ang II concentrations. The relationship was then fitted to the Hill equation with the Marquardt-Levenberg algorithm:

%NP/NP_o=100x{1+([Ang II]/IC₅₀)ⁿ}^-1, where IC₅₀ is the half-maximal concentration for inhibition of Ang II, and n is the Hill coefficient. Numerical data are given as mean±SEM.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Ang II Inhibition of Whole-Cell I_KATP
Fig 1 represents the action of Ang II on whole-cell currents. Exposure of a ventricular myocyte pretreated with 2-deoxyglucose (5.5 mmol/L) to glucose-free Tyrode's medium containing 1 mmol/L CN^- produced an outward current at a -40 mV holding potential within 2 to 3 minutes (Fig 1A, a to b). Fig 1B shows the quasi-instantaneous current-voltage relationships obtained through ramp-pulse stimulation. Fig 1B (a through f) in the current-voltage relations corresponds to the chart in Fig 1A.

View larger version (28K): [in this window] [in a new window]   Figure 1. Effect of Ang II on IKATP. A, Chart recording of IKATP activated by metabolic poisoning with 5.5 mmol/L 2-deoxyglucose and 1 mmol/L CN- at a holding potential of -40 mV. Horizontal arrow on the left of the trace shows the zero current level. Bars above the trace indicate the application of various test solutions. B, Current-voltage curve at the point of each vertical arrow (a through f ) (top). C, Difference currents obtained from b to a, c to d, and e to f (middle). a, Control; b, currents activated by 1 mmol/L cyanide; c, application of glucose; d, inhibitory effect of glucose; e, application of Ang II (1 nmol/L); f, inhibitory effect of Ang II.

The conductance induced by the metabolic inhibition (b to a), obtained as the difference current shown in Fig 1C (i), indicated a marked outward rectification with a reversal of -80 mV, which is close to the estimated equilibrium potential for K⁺ under the present recording conditions (E_K=-85 mV).

The extracellular addition of 5.5 mmol/L glucose and the removal of CN^- partially reduced the channel conductance (Fig 1A [d], 1B [ii], and 1C [ii]), presumably through the relieving metabolic suppression. This glucose-sensitive current had a reversal potential of -78 mV, which is also close to the E_K. These findings suggested that the metabolically evoked current was carried by K_ATP.

Subsequent exposure of the myocyte to Ang II (1 nmol/L) suppressed the conductance in a reversible and repeated manner (Fig 1A [f], 1B [iii], and 1C [iii]). The Ang II–blockable conductance reversed at -80 mV, which is again close to the E_K. The findings were similar in three other experiments, suggesting that Ang II blocks K_ATP.

Ang II–Dependent Closure of Single K_ATP in the Cell-Attached Experiments
To further examine the mechanism that controls this Ang II–dependent inhibition, we recorded the current of single K_ATP in the cell-attached mode activated by metabolic stress. Exposure of a cell preincubated with 2-deoxyglucose (5.5 mmol/L) to NaCN (1 mmol/L) produced outward single-channel openings at a 0 mV holding potential within 10 to 15 minutes, as shown in Fig 2A. Glibenclamide (1 µmol/L) applied outside the patch pipette completely inhibited the channel currents thus activated in a reversible manner (Fig 2A).

View larger version (22K): [in this window] [in a new window]   Figure 2. Effect of glibenclamide and Ang II on KATP in single-channel recordings. A, Single-channel currents activated by metabolic poisoning were reversibly inhibited by 1 µmol/L gli-benclamide. B, Unitary current-potential relationships. C, Concentration-dependent inhibition of KATP by Ang II. D, Concentration-inhibition relation between [Ang II] and %NP/NPo of KATP. Symbols indicate mean values; bars, SEM values; and numbers in parentheses, number of observations. Data were fitted to the Hill equation: %NP/NPo=100/{1+([Ang II]/Kd)n}; where [Ang II] is the concentration of Ang II, and n is the Hill coefficient.

Fig 2B reveals unitary current-membrane potential relationships obtained from four experiments. The reversal potential was -84.7±2.6 mV, and the single-channel conductance was 19.8±0.8 pS. These findings corresponded well with those previously reported²⁶ and identified channels opened by metabolic stress as single K_ATP.

Subnanomolar Ang II outside the patch pipette inhibited the K_ATP activity in a reversible manner (Fig 2C). The inhibitory action of Ang II was concentration dependent, and Fig 2D summarizes the data obtained from 29 observations. The inhibitory effects of Ang II appeared to be saturated at the concentration over 0.1 nmol/L. The smooth curve in the graph is the best fit to the Hill equation, with an IC₅₀ value of 3.2±0.5 pmol/L and a Hill coefficient of 1.3±0.3.

Ang II Acts Through the AT₁ Receptor
On excising the cell-attached membrane patch in the standard 150 mmol/L K⁺ solution without ATP and CN^-, the K_ATP activity suddenly increases. After the channel activity attained a steady state in the inside-out patch (Fig 3A), Ang II (1 nmol/L) failed to inhibit K_ATP. But glibenclamide (1 µmol/L) shut down them, suggesting that Ang II acts on K_ATP from outside the cell membrane, probably through the receptor.

View larger version (25K): [in this window] [in a new window]   Figure 3. Effects of Ang II on KATP in the presence of the AT1 and AT2 receptor antagonist. A, Chart of single-channel recording from an inside-out patch. Bars above the chart indicate applications of a given agent from the internal side of the membrane. B and C, In the cell-attached mode, KATP were opened by metabolic stress. Bars indicate applications of agents to the outside of the pipette. Recording modes are schematically shown to the left of each chart.

The experimental condition in Fig. 3B and 3C was the cell-attached mode, as indicated schematically in the inset. Two types of Ang II receptors are known to couple to various signal transduction pathways.^{10 11} We investigated which type of receptor is involved in the Ang II–induced inhibition of K_ATP by using CV11974 (100 nmol/L), an AT₁ receptor–selective antagonist,²⁷ and PD123319 (100 nmol/L), an AT₂ receptor–selective antagonist.²⁸ These compounds alone did not prevent the channel activation induced by metabolic stress. CV11974 (100 nmol/L) antagonized the inhibitory action of Ang II. After washing out the antagonist, the same concentration of Ang II, however, closed the channels (Fig 3B). In contrast, PD123319 (100 nmol/L) failed to affect the inhibitory effect of Ang II (Fig 3C). Thus, AT₁ receptors are involved in this Ang II action.

Intracellular Mechanism of Ang II–Mediated Inhibition of K_ATP
Because Ang II was capable to suppress the K_ATP when applied to the outside of the pipette in the cell-attached condition, cytosolic soluble second messenger(s) may be involved. As for a candidate for such messenger, we sought whether Ang II affects subsarcolemmal ATP concentration and thereby the channel activity. To examine the intracellular signal transduction pathway, the open-cell–attached mode was attained through the brief application (usually 3 minutes) of bathing solution (150 mmol/L KCl, 10 mmol/L HEPES, 1 mmol/L ATP, and 1 mmol/L EGTA) containing streptolysin-O (0.08 U/mL) after the formation of the cell-attached mode. As represented in Fig 4Aa, decreasing ATP in the bathing solution from 1 to 0.1 mmol/L typically opened K_ATP. The subsequent application of Ang II (1 nmol/L; outside of the pipette, indicated by a horizontal bar above the chart) was no longer inhibitory. Resumption of the extracellular ATP concentration to 1 mmol/L reversibly blocked the channel activation, although recovery was partial in this particular experiment (to the right of the chart).

View larger version (25K): [in this window] [in a new window]   Figure 4. Lack of Ang II action on KATP in the open-cell–attached mode. Aa, In the open-cell–attached mode referred, KATP were activated by lowering the extracellular ATP concentration. Bars above the chart indicate applications of agents. Ab, In the cell-attached mode, KATP were activated by metabolic suppression. The extracellular application of ATP (1 mmol/L) showed no effect. B, Ang II lost its inhibitory action on KATP activity induced by cromakalim in the cell-attached mode.

In contrast, when the channels were activated in the cell-attached mode by metabolic stress (Fig 4Ab), ATP (1 mmol/L) applied outside the pipette did not affect the channel activity (to the left of the chart), suggesting that the cell membrane remained intact in these recording conditions. However, extracellular (outside the pipette) Ang II (1 nmol/L) reversibly inhibited the channel activity. Thus, the level of subsarcolemmal [ATP] may be altered during the Ang II–mediated inhibition of K_ATP.

This notion was also supported by the experimental result using cromakalim, a K_ATP opener, in the cell-attached mode. After cromakalim (100 µmol/L) activated K_ATP without introducing metabolic stress, Ang II (100 nmol/L) could no longer inhibit K_ATP activity, but glibenclamide reversibly blocked this activity (Fig 4B). A similar lack of inhibitory action of Ang II was consistently observed in five other myocytes. Taken together, the increase in subsarcolemmal [ATP] may be involved in the Ang II–induced inhibition of the channel because Ang II lost its action (1) when [ATP] can be controlled by an exogenous ATP (open-cell–attached mode) or (2) when the channels are opened by cromakalim, not by the [ATP] reduction primarily induced by metabolic stress.

Although [ATP] appears to be the second messenger of Ang II–mediated inhibition of the channels, the actual linker between Ang II receptors and [ATP] remains unknown. Ang II receptors belong to the G protein-coupled receptor superfamily with seven-transmembrane segments.^{10 11} Because the AT₁ type of Ang II receptors was shown to negatively couple to adenylate cyclase via PTX-sensitive G proteins in the heart,^{29 30} we examined whether this type of G protein coupled to Ang II–mediated inhibition of K_ATP channels is sensitive to PTX by preincubating the myocytes with the toxin. After exposure to PTX,³¹ Ang II (100 nmol/L) failed to close the channels that had been activated by metabolic inhibition (Fig 5A). However, an extracellular application of glibenclamide (100 nmol/L) completely suppressed it. This finding was consistently observed in five other cells, suggesting that PTX-sensitive G proteins mediate the Ang II action.

View larger version (33K): [in this window] [in a new window]   Figure 5. Effects of PTX and inhibition of Na+,K+-ATPase. A, The myocyte was preincubated with PTX. B, Inhibitory effects of ouabain (10 µmol/L) on KATP channels. C, Inhibitory effects of ouabain (100 µmol/L) disappeared in the open-cell–attached mode attained by prior exposure to streptolysin-O (0.08 U/mL). D, Inhibitory effects of Ang II on KATP despite the continued presence of ouabain (100 µmol/L).

More recently, Priebe et al³² have shown that the digitalis-induced suppression of Na⁺,K⁺-ATPase reversibly and ATP-dependently closed K_ATP by minimizing the consumption of subsarcolemmal ATP by the enzyme in guinea pig ventricular myocytes. In the last series of experiments, we therefore tested the action of ouabain and Ang II on K_ATP activated by metabolic stress. Ouabain (10 µmol/L) applied outside of the patch electrode in the absence of extracellular ATP (under cell-attached condition) virtually closed the channel in a reversible and repeated manner (Fig 5B). Because this type of inhibition was not evident in the open-cell–attached mode attained by streptolysin-O in the absence of extracellular ATP (Fig 5C), it was likely that ouabain indeed acts by modulating [ATP] via the inhibition of Na⁺,K⁺-ATPase and the consequent increase in [ATP].

In the prolonged presence of ouabain (100 µmol/L), K_ATP began to reopen, reflecting the [ATP] decrease at ATP binding sites of channels and/or associated molecules (Fig 5D). After resumption of channel activities, Ang II outside the pipette electrode reversibly suppressed the channel activity. It was therefore concluded that Na⁺,K⁺-ATPase is not involved in Ang II–mediated inhibition of the channel.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Intracellular ATP-Dependent Regulation of K_ATP Activity
Fig 6 illustrates our proposed mechanism for the Ang II–mediated modulation of K_ATP. Ang II appeared to suppress the K_ATP activity through an increase in the subsarcolemmal [ATP] that resulted from the suppression of adenylate cyclase via an AT₁ receptor/PTX-sensitive G protein.

View larger version (17K): [in this window] [in a new window]   Figure 6. Intracellular [ATP]-dependent regulation of KATP. ATP in the square indicates the subsarcolemmal ATP; broken lines, inhibition of signal transduction.

In general, Ang II stimulation facilitates membrane phosphoinositide hydrolysis by phospholipase C, thereby generating inositol-1,4,5-triphosphate and diacylglycerol and coupling to PKC activation. The involvement of PKC pathway for the regulation of K_ATP, however, remained controversial.^{33 34 35 36 37} The PKC-dependent change of K_ATP activity was similarly seen in both human and rabbit cardiocytes.^{34 36 37}

Using the excised inside-out patch method in rabbit ventricular myocytes, Light et al^{34 37} showed that PKC inhibits K_ATP at a low cytoplasmic ATP concentration but activates them in the physiological (mmol/L) level of ATP and thereby alters the sensitivity of the channel to ATP. In another set of inside-out patch experiments, we therefore tested whether Ang II enhances the channel sensitivity to blockade by ATP. The inclusion of 1 nmol/L Ang II in pipettes did not produce a significant difference in ATP-dependent closure of the channel: percent inhibition by 10 µmol/L ATP at 0 mV holding potential was 15.1±1.2% in control and 14.5±3.2% in Ang II, and that by 50 µmol/L was 66.0±2.8 and 56.1±3.5, respectively. It was therefore concluded that Ang II did not change the channel sensitivity to ATP in the present study.

The sensitivity to PTX, shown here, did not exclude the involvement of this pathway in Ang II–induced inhibition of K_ATP channels because some G proteins coupling to this system are PTX sensitive.¹⁸ This was, however, unlikely because the activities of phospholipase C and PKC are highly Ca²⁺ sensitive and because under our experimental condition, with the whole-cell mode (Fig 1), intracellular Ca²⁺ was tightly chelated by very low concentrations (0.1 to 1 nmol/L) of a Ca²⁺-free, K⁺-rich pipette solution containing 10 mmol/L EGTA.

In contrast, using a nystatin-perforated whole-cell technique to cat atrial cells, Wang and Lipsius³³ demonstrated that when an atrial myocyte is consecutively exposed twice to acetylcholine separated by a recovery interval, the second exposure elicits a larger increase in glibenclamide-blockable K⁺ conductance than the first. A PKC-dependent pathway appeared to be involved in this acetylcholine-induced facilitation because the K⁺ current increase was abolished through depletion of the sarcoplasmic reticulum Ca²⁺ stores with 1 µmol/L ryanodine or 10 mmol/L caffeine, intracellular dialysis with 10 mmol/L EGTA, or inhibition of PKC.

The Ang II–induced inhibition of K_ATP appeared to depend on [ATP]. This notion was supported by the experimental finding that introduction of the open-cell–attached mode completely abolished any inhibitory action of Ang II (Fig 4). In this mode, through tiny membrane holes, [ATP] can be clamped at a desired level by bathing extracellular solutions with a given ATP concentration. Thus, under the condition in which [ATP] cannot be readily altered, Ang II was without effect. Similarly, when K_ATP were activated by cromakalim, Ang II was unable to inhibit the channel activity.

Subsarcolemmal [ATP] can be altered as a result of the activation or suppression of adenylate cyclase activity, as previously shown in cat cardiocytes by Schackow and Ten Eick.³⁸ The opening of K_ATP was accelerated by ß-adrenoceptor stimulation but suppressed by adenosine. The mechanism of this ß-adrenoceptor–mediated stimulatory action was contributed to the decrease in [ATP] due to the consumption of endogenous ATP by the activation of the membrane-bound enzyme adenylate cyclase via G_s in the vicinity of the membrane.

Myocardial AT₁ receptors were shown to negatively couple to adenylate cyclase via PTX-sensitive G proteins. First, with the application of radioimmunoassay to isolated sarcolemmal fractions from rat heart cells, Anand-Srivastava demonstrated²⁹ that Ang II negatively couples to adenylate cyclase. Second, with patch-clamp techniques, Habuchi et al³⁹ have shown that basal Ca²⁺ channel currents in rabbit nodal cells are concentration-dependently inhibited by Ang II via AT₁ receptors/PTX-sensitive G proteins. Third, with the use of radioimmunoassay and patch-clamp techniques, we also demonstrated that Ang II stimulation negatively couples to the adenylate cyclase in the same preparation (ie, guinea pig ventricular myocytes).³¹ Thus, Ang II–induced inhibition of K_ATP can be explained, at least in part, by an increase in [ATP] that resulted from the suppression of adenylate cyclase via an AT₁ receptor/PTX-sensitive G protein.

The activity of Na⁺,K⁺-ATPase was also shown to regulate the K_ATP activity in a similar manner by [ATP]. Priebe et al³² have shown that the inhibition of Na⁺,K⁺-ATPase by digitalis reversibly causes ATP-dependent closure of the channels by minimizing the consumption of [ATP] by membrane Na⁺,K⁺-ATPase in guinea pig ventricular myocytes. Our results shown in Fig 5B agree with their findings. Because the inhibitory action of Ang II was not affected in the continued presence of ouabain (100 µmol/L; Fig 5C), it is unlikely that Ang II acts through the inhibition of Na⁺,K⁺-ATPase.

Concentration gradients for intracellular substances such as Na⁺, Ca²⁺, and ATP are noted in the vicinity of the cell membrane ("fuzzy space"⁴⁰ ). There may be two factors that determine the [ATP] gradient: (1) the nonhomogeneous distribution of mitochondria and (2) glycolysis-dependent ATP production. Using saponin-perforated open-cell–attached, patch-clamp techniques in guinea pig ventricular myocytes, Weiss and Lamp^{41 42} found that ATP derived from glycolysis rather than oxidative phosphorylation more effectively prevents K_ATP activation. It was suggested that this phenomenon occurs because glycolytic enzymes are located in the vicinity of the channels. Therefore, the presence of a dynamic [ATP] gradient supports our finding that modulation of membrane enzyme activities that consume ATP can in turn change [ATP] and, therefore, the channel activity.

Physiological and Clinical Implications of Ang II Inhibition on K_ATP and Experimental Limitations
The plasma concentration of Ang II reportedly ranges between 5 and 50 pmol/L in humans,⁴³ and Ang II secretion is elevated in various diseases with metabolic stress.⁶ The ventricular remodeling and compensatory hypertrophy seen after myocardial infarction are related to a local increase in the production of Ang II.⁴⁴ It is therefore likely that the local Ang II level would be higher than that in plasma under pathological conditions in which K_ATP begin to open.^{21 22 45}

In this study, Ang II suppressed K_ATP in both whole-cell and cell-attached modes. However, the concentration level for the inhibitory action of Ang II differed depending on the experimental mode (1 nmol/L in the whole-cell mode and 0.01 nmol/L in the cell-attached mode). This discrepancy in concentration may be attributed to (1) the concentration clamp as a result of cell perfusion with a pipette solution containing 0.1 mmol/L ATP in the conventional whole-cell mode because the subsarcolemmal [ATP] should be influenced by exogenous intrapipette ATP. Indeed, when we used lower-resistance, wide-tipped pipettes, Ang II action on the whole-cell K_ATP became smaller and even faded (data not shown), suggesting that a smooth intracellular dialysis with exogenous ATP prevents an Ang II–induced change in [ATP] that affects the K_ATP conductance. (2) In the cell-attached mode, [ATP] level could be monitored by the activity of the channel in a small cell membrane in the tip of the patch pipette, but Ang II was applied to the outside of the pipette (whole myocyte), so an Ang II–induced rise in [ATP] may influence the level of [ATP] monitored at a very tiny cell membrane sooner at the site of channel monitoring (beneath the pipette). Therefore, both modes for K_ATP currents had experimental limitations in the present study.

Antagonization of Ang II action by ACE inhibitors improves the symptoms of congestive heart failure and reduces reperfusion injury and arrhythmias.^{46 47 48} Using the rat model, Linz et al⁶ demonstrated that ACE inhibitors reduce both ischemic areas at risk and zones of infarcted muscle. The inhibitory pathway of K_ATP may be one of several actions of Ang II and in part account for the cardioprotective actions of ACE inhibitors. Ang II receptor antagonists may also provide another therapeutic modality because receptor antagonism is more potent than ACE inhibitors, which cannot inhibit Ang II generation by nonrenin enzymes.^{49 50}

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II AT1 receptor = angiotensin II type 1 receptor IKATP = ATP-sensitive K+ channel current(s) KATP = ATP-sensitive K+ channel(s) PKC = protein kinase C PTX = pertussis toxin RAS = renin-angiotensin-aldosterone system

	Acknowledgments

 
This work was supported by Grants-in-Aid on Priority Areas of "Channel-Transporter Correlation" from the Japan Ministry of Education, Science and Culture. C.A.A. is the recipient of a scholarship from the Japan Ministry of Education, Science and Culture.

Received March 12, 1997; revision received May 19, 1997; accepted May 28, 1997.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Dempsey PJ, McCallum ZT, Kent KM, Cooper T. Direct myocardial effects of angiotensin II. Am J Physiol. 1971;220:477-481.

2. Freer RJ, Pappano AJ, Peach MJ, Bing KT, McLean MJ, Vogel S, Sperelakis N. Mechanism for the positive inotropic effects of angiotensin II on isolated cardiac muscle. Circ Rec. 1976;39:178-183.[Abstract/Free Full Text]

3. Regoli D, Park WK, Rioux F. Pharmacology of angiotensin. Pharmacol Rev. 1974;26:69-123.[Free Full Text]

4. Peach MJ. Renin-angiotensin system: biochemistry and mechanisms of action. Physiol Rev. 1977;57:313-370.[Free Full Text]

5. Barrett PQ, Bollag WB, Isales CM, Isales CM, McCarthy RT, Rasmussen H. Role of calcium in angiotensin II-mediated aldosterone secretion. Endocr Rev. 1989;10:496-518.[Abstract/Free Full Text]

6. Linz W, Scholkens BA, Han JF. Beneficial effects of the converting enzyme inhibitor ramipril in ischemic rat heart. J Cardiovasc Pharmacol. 1986;8(suppl 10):S91-S99.

7. Peach MJ. Renin-angiotensin system: mechanism of action. Biochem Physiol Rev. 1981;57:313-370.

8. Rogers TB, Gaa ST, Allen IS. Identification and characterization of functional angiotensin-II receptors in cultured heart myocytes. J Pharmacol Exp Ther. 1986;236:438-444.[Abstract/Free Full Text]

9. Rogers TB. High affinity angiotensin II receptors in myocardial sarcolemmal membranes. J Biol Chem. 1984;259:8106-8113.[Abstract/Free Full Text]

10. Murphy TJ, Alexander RW, Kathy KG. Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor. Nature. 1991;351,16:233-236.

11. Sasaki K, Yamano Y, Smriti B. Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type-1 receptor. Nature. 1991;351,16:230-232.

12. Dzau VJ, Ingelfinger Y, Pratt RE, Ellison KE. Identification of renin and angiotensinogen RNA sequences in mouse and rat brains. Hypertension. 1986;8:544-558.[Abstract/Free Full Text]

13. Baker KM, Singer HA. Identification and characterization of guinea pig angiotensin-II ventricular and atrial receptors: coupling to inositol phosphate production. Circ Rec. 1988;62:896-904.[Abstract/Free Full Text]

14. Baker KM, Aceto JA. Characterization of avian angiotensin-II cardiac receptors: coupling to mechanical activity and phosphoinositide receptors. J Mol Cell Cardiol. 1989;21:375-382.[Medline] [Order article via Infotrieve]

15. Bahinski A, Nairn AC, Greengard P, Gadsby DC. Chloride conductance regulated by cyclic AMP-dependent protein kinase in cardiac myocytes. Nature. 1989;340:718-721.[Medline] [Order article via Infotrieve]

16. James AF, Xie L-H, Fujitani Y, Hayashi S, Horie M. Inhibition of the cardiac protein kinase A-dependent chloride conductance by endothelin-1. Nature. 1994;370:297-300.[Medline] [Order article via Infotrieve]

17. Sadoshima JI, Izumo S. Signal transduction pathways of angiotensin II-induced c-fos gene expression in cardiac myocytes in vitro. Circ Res. 1993;73:424-438.[Abstract/Free Full Text]

18. Baker KM, Booz GW, Dostal DE. Cardiac actions of angiotensin II: role of an intracardiac renin-angiotensin system. Annu Rev Physiol. 1992;54:227-241.[Medline] [Order article via Infotrieve]

19. Noma A. ATP-regulated K⁺ channels in cardiac muscle. Nature. 1983;305:147-148.[Medline] [Order article via Infotrieve]

20. Nabika T, Velletri PA, Lovenberg W. Increase in cytosolic calcium and phosphoinositide metabolism induced by angiotensin II and [Arg]vasopressin in vascular smooth muscle cells. J Biol Chem. 1985;260:4661-4670.[Abstract/Free Full Text]

21. Nichols CG, Lederer WJ. Adenosine triphosphate-sensitive potassium channels in the cardiovascular system. Am J Physiol. 1991;261:H1675-H1686.[Abstract/Free Full Text]

22. Gross GJ, Auchanpach JA. Blockade of ATP-sensitive potassium channels prevents myocardial preconditioning in dogs. Circ Res. 1992;70:223-233.[Abstract/Free Full Text]

23. Grover GJ, Sleph PG, Dzwonczyk S. Role of myocardial ATP-sensitive potassium channels in mediating preconditioning in the dog heart and their possible interaction with adenosine A₁ receptors. Circulation. 1992;86:1310-1316.[Abstract/Free Full Text]

24. Isenberg G, Klöckner U. Calcium tolerant ventricular myocytes prepared by preincubation in a `KB medium.' Pflugers Arch. 1982;395:6-18.[Medline] [Order article via Infotrieve]

25. Tsuura Y, Ishida H, Hayashi S, Sakamoto K, Horie M, Seino Y. Nitric oxide opens ATP-sensitive K⁺ channels through suppression of phosphofructokinase activity and inhibits glucose-induced insulin release in pancreatic ß cells. J Gen Physiol. 1994;104:1079-1099.[Abstract/Free Full Text]

26. Ashcroft FM. Adenosine 5'-triphosphate-sensitive potassium channels. Annu Rev Neurosci. 1988;11:97-118.[Medline] [Order article via Infotrieve]

27. Noda M, Shibouta Y, Inada Y, Ojima M, Wada T, Sanada T, Kubo K, Kohara Y, Naka T, Nishikawa K. Inhibition of rabbit aortic angiotensin II (A-II) receptor by CV-11974, a new nonpeptide A-II antagonist. Biochem Pharmacol. 1993;46:311-318.[Medline] [Order article via Infotrieve]

28. Dudley DT, Panek RL, Major TC, Lu GH, Bruns RF, Klinkefus BA, Hodges JC, Weishaar RE. Subclasses of angiotensin II binding sites and their functional significance. Mol Pharmacol. 1990;38:370-377.[Abstract]

29. Anand-Srivastava MB. Angiotensin II receptors negatively coupled to adenylate cyclase in rat myocardial sarcolemma: involvement of inhibitory guanine nucleotide regulatory protein. Biochem Pharmacol. 1989;38:489-496.[Medline] [Order article via Infotrieve]

30. Obayashi K, Horie M, Xie L-H, Tsuchiya K, Kubota A, Ishida H, Sasayama S. Angiotensin-II inhibits protein kinase A–dependent Cl^- conductance in heart via PTX-sensitive G proteins. Circulation. 1997;95:197-204.[Abstract/Free Full Text]

31. Ui M. Pertussis toxin as a valuable probe for G-protein involvement in signal transduction. In: Moss J, Vaughan M, eds. ADP-Ribosylation Toxins and G Proteins: Insight Into Signal Transduction. Washington, DC: American Society for Microbiology; 1990:45-77.

32. Priebe L, Friedrich M, Benndorf K. Influence of Na⁺-K⁺-pump action on the activity of K_ATP channels in isolated guinea pig heart cells. Biophysical J. 1995;68:A251. Abstract.

33. Wang YG, Lipsius SL. Acetylcholine activates a glibenclamide-sensitive K⁺ current in cat atrial myocytes. Am J Physiol. 1995;268:H1322-H1334.[Abstract/Free Full Text]

34. Light PE, Allen BG, Walsh MP, French RJ. Regulation of adenosine triphosphate-sensitive potassium channels from rabbit ventricular myocytes by protein kinase C and type 2A protein phosphatase. Biochemistry. 1995;34:7252-7257.[Medline] [Order article via Infotrieve]

35. Baker KM, Singer HA, Aceto JF. Angiotensin II receptor- mediated stimulation of cytosolic-free calcium and inositol phosphates in chick myocytes. J Pharmacol Exp Ther. 1989;251:578-585.[Abstract/Free Full Text]

36. Hu K, Duan D, Li GR, Nattel S. Protein kinase C activates ATP-sensitive K⁺ current in human and rabbit ventricular myocytes. Circ Res. 1996;78:492-498.[Abstract/Free Full Text]

37. Light PE, Sabir AA, Allen BG, Walsh MP, French RJ. Protein kinase C-induced changes in the stoichiometry of ATP binding activate cardiac ATP-sensitive K⁺ channels: a possible mechanistic link to ischemic preconditioning. Circ Res. 1996;79:399-406.[Abstract/Free Full Text]

38. Schackow, TE, Ten Eick RE. Enhancement of ATP-sensitive potassium current in cat ventricular myocytes by ß-adrenoreceptor stimulation. J Physiol. 1994;474:131-145.[Abstract/Free Full Text]

39. Habuchi H, Lu L-L, Morikawa J, Yoshimura M. Angiotensin II inhibition of L-type Ca²⁺ current in sinoatrial node cells in rabbits. Am J Physiol. 1995;268(Heart Circ Physiol. 37):H1053-H1060.

40. Carmeliet E. A fuzzy subsarcolemmal space for intracellular Na⁺ in cardiac cells? Cardiovasc Res. 1992;26:433-442.[Medline] [Order article via Infotrieve]

41. Weiss JN, Lamp ST. Glycolysis preferentially inhibits ATP-sensitive K⁺ channels in isolated guinea-pig myocytes. Science. 1987;238:67-69.[Abstract/Free Full Text]

42. Weiss JN, Lamp ST. Cardiac ATP-sensitive K⁺ channels. J Gen Physiol. 1989;94:911-935.[Abstract/Free Full Text]

43. Düsterdieck G, McElwee G. Estimation of angiotensin II concentration in human plasma by radioimmunoassay: some applications to physiological and clinical states. Eur J Clin Invest. 1971;2:32-38.[Medline] [Order article via Infotrieve]

44. Carroll EP, Janicki JS, Pick R, Weber KT. Myocardial stiffness and reparative fibrosis following coronary embolization in the rat. Cardiovasc Res. 1989;23:655-661.[Medline] [Order article via Infotrieve]

45. Masaki T, Yanagisawa Y, Goto K. Physiology and pharmacology of endothelin. Med Res Rev. 1992;12:391-421.[Medline] [Order article via Infotrieve]

46. Thous DW, Baker KM, Ayers CR, Vaughan ED, Carley RM. Acute and chronic effect of captopril in hypertensive patients. Hypertension. 1980;2:567-575.[Free Full Text]

47. Elfellah MS, Oglive RI. Effect of vasodilator drugs on coronary occlusion and reperfusion arrhythmias in anaesthetized dogs. J Cardiovasc Pharmacol. 1985;7:826-832.[Medline] [Order article via Infotrieve]

48. Van Gilst WH, De Graeff PA, Wesseling H, de Langen CDJ. Reduction of reperfusion arrhythmias in the ischemic isolated rat heart by angiotensin converting enzyme inhibitors: a comparison of captopril, enalapril and HOE 498. J Cardiovasc Pharmacol. 1986;8:722-728.[Medline] [Order article via Infotrieve]

49. Hirakata H, Found-Tarazi FM, Bumpus FM, Khosla M, Healy B. Angiotensins and the failing heart: enhanced positive inotropic response to angiotensin-I in cardiomyopathic hamster heart in the presence of captopril. Circ Res. 1990;66:891-899.[Abstract/Free Full Text]

50. Urata H, Kinosita A, Misono KS, Bumpus FM, Fusain A. Identification of a highly specific chymase as the major angiotensin-II-forming enzyme in the human heart. J Biol Chem. 1990;265:22348-22357.[Abstract/Free Full Text]

This article has been cited by other articles:

				D. Ai, W. Pang, N. Li, M. Xu, P. D. Jones, J. Yang, Y. Zhang, N. Chiamvimonvat, J. Y.-J. Shyy, B. D. Hammock, et al. Soluble epoxide hydrolase plays an essential role in angiotensin II-induced cardiac hypertrophy PNAS, January 13, 2009; 106(2): 564 - 569. [Abstract] [Full Text] [PDF]

				J. F. Buckley, M. Singer, and L. H. Clapp Role of KATP channels in sepsis Cardiovasc Res, November 1, 2006; 72(2): 220 - 230. [Abstract] [Full Text] [PDF]

				P. Dhar-Chowdhury, M. D. Harrell, S. Y. Han, D. Jankowska, L. Parachuru, A. Morrissey, S. Srivastava, W. Liu, B. Malester, H. Yoshida, et al. The Glycolytic Enzymes, Glyceraldehyde-3-phosphate Dehydrogenase, Triose-phosphate Isomerase, and Pyruvate Kinase Are Components of the KATP Channel Macromolecular Complex and Regulate Its Function J. Biol. Chem., November 18, 2005; 280(46): 38464 - 38470. [Abstract] [Full Text] [PDF]

				T. Haruna, H. Yoshida, T. Y. Nakamura, L.-H. Xie, H. Otani, T. Ninomiya, M. Takano, W. A. Coetzee, and M. Horie {alpha}1-Adrenoceptor-Mediated Breakdown of Phosphatidylinositol 4,5-Bisphosphate Inhibits Pinacidil-Activated ATP-Sensitive K+ Currents in Rat Ventricular Myocytes Circ. Res., August 9, 2002; 91(3): 232 - 239. [Abstract] [Full Text] [PDF]

				H. Yu, J. Gao, H. Wang, R. Wymore, S. Steinberg, D. McKinnon, M. R. Rosen, and I. S. Cohen Effects of the Renin-Angiotensin System on the Current Ito in Epicardial and Endocardial Ventricular Myocytes From the Canine Heart Circ. Res., May 26, 2000; 86(10): 1062 - 1068. [Abstract] [Full Text] [PDF]

				E. Carmeliet Cardiac Ionic Currents and Acute Ischemia: From Channels to Arrhythmias Physiol Rev, July 1, 1999; 79(3): 917 - 1017. [Abstract] [Full Text] [PDF]

				T. Haruna, M. Horie, I. Kouchi, R. Nawada, K. Tsuchiya, M. Akao, H. Otani, T. Murakami, and S. Sasayama Coordinate Interaction Between ATP-Sensitive K+ Channel and Na+,K+-ATPase Modulates Ischemic Preconditioning Circulation, December 22, 1998; 98(25): 2905 - 2910. [Abstract] [Full Text] [PDF]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tsuchiya, K. Articles by Sasayama, S. Search for Related Content PubMed PubMed Citation Articles by Tsuchiya, K. Articles by Sasayama, S.