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Circulation. 1997;95:197-204

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(Circulation. 1997;95:197-204.)
© 1997 American Heart Association, Inc.

Articles

Angiotensin II Inhibits Protein Kinase A–Dependent Chloride Conductance in Heart via Pertussis Toxin–Sensitive G Proteins

Kazuhiko Obayashi, MD; Minoru Horie, MD, PhD; Lai-Hua Xie, MS; Kunihiko Tsuchiya, MD; Akira Kubota, MD; Hitoshi Ishida, MD, PhD; Shigetake Sasayama, MD, PhD

the Department of Cardiovascular Medicine and Department of Metabolism and Clinical Nutrition (A.K., H.I.), Faculty of Medicine, Kyoto University, Japan.

Correspondence to Minoru Horie, Division of Cardiac Electrophysiology, Department of Cardiovascular Medicine, Faculty of Medicine, Kyoto University, Shogoin, Sakyo-ku, Kyoto 606-01, Japan. E-mail horie@kuhp.kyoto-u.ac.jp.


*    Abstract
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Background Angiotensin II receptors are reported to be abundant in the guinea pig ventricle; their coupling to adenylate cyclase in the heart, however, remains controversial. Therefore, we investigated the effect of angiotensin II on Cl- conductance activated by cAMP-dependent protein kinase.

Methods and Results After minimizing the contribution of other ionic currents, exposure of single guinea pig ventricular cells to isoproterenol (40 to 50 nmol/L; 36°C) elicited a typical protein kinase A–dependent Cl- conductance. Subsequent application of angiotensin II reduced the isoproterenol-induced conductance with an IC50 of 0.24±0.08 nmol/L. Angiotensin II also inhibited the Cl- currents, which were activated through stimulation of adenylate cyclase by forskolin and histamine receptors. CV-11974 (1 µmol/L), an antagonist selective for the angiotensin type 1 receptor, prevented the effect of angiotensin II. Angiotensin II did not inhibit the current that had been persistently activated by intracellular GTP{gamma}S (100 µmol/L), a nonhydrolyzable guanine nucleotide, plus isoproterenol. In addition, prior incubation of myocytes with pertussis toxin prevented the angiotensin II inhibitory action. Cl- conductance, when activated directly by intracellular dialysis with cAMP (1 mmol/L), was not affected by angiotensin II. Radioimmunologic measurement of cellular cAMP in the dissociated myocytes showed that angiotensin II inhibited the isoproterenol-induced increase of cAMP.

Conclusions Angiotensin II receptors negatively couple to adenylate cyclase via pertussis toxin–sensitive G proteins, thereby inhibiting cardiac protein kinase A–dependent Cl- conductance.


Key Words: angiotensin • signal transduction • ions • heart failure


*    Introduction
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During the past decade, one of the most remarkable advances in heart failure therapy has come from the large-scale randomized clinical trials demonstrating the beneficial effect of ACE inhibitors in reducing morbidity and mortality, thereby highlighting pathophysiological roles of the renin-angiotensin system.1 2 3 4 Tissue levels of A-II, a principal effector of the renin-angiotensin system, are known to be increased without elevating plasma renin levels, particularly under pathological conditions. Receptor-binding studies revealed that A-II receptors are expressed abundantly in the myocardium.5 6 7 8 The octapeptide hormone A-II exerts a positive inotropic action9 10 11 and promotes myocardial hypertrophy after acute myocardial infarction12 13 14 and in hypertensive rat models.15 16 Thus, the previous concept of the renin-angiotensin system has been expanded: A-II may act as an autocrine and/or paracrine peptide hormone in heart (for review, see References 17 and 18).

A-II receptors have been shown to couple to various signal transduction pathways in the myocardium that involve an increase of cellular second messengers such as inositol 1,4,5-triphosphate, phosphatidic acid, diacylglycerol, and arachidonic acid, thereby activating the PKC pathway.18 19 20 In contrast, the coupling to the PKA system remains controversial, with some reports of no coupling to adenylate cyclase5 20 and others of negative coupling through PTX-sensitive G proteins.21 In other organs, such as the kidney,22 adrenal cortex,23 24 liver,25 and vascular smooth muscle,26 inhibition of adenylate cyclase by A-II has been demonstrated.

The objective of the present study was to examine whether A-II was linked to the adenylate cyclase/PKA system in the guinea pig heart, in which A-II receptors have been reported to be enriched.7 We measured PKA-dependent Cl- conductance27 28 29 30 31 32 as well as the intracellular level of cAMP with RIA. We found that A-II reduces Cl- conductance by inhibiting adenylate cyclase via AT1 receptors/PTX-sensitive G proteins.


*    Methods
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Preparation of Single Myocytes
Ventricular myocytes were prepared by enzymatic digestion of adult guinea pig heart with Langendorff's perfusion of collagenase, as described previously.31 Briefly, adult guinea pigs were fully anesthetized with pentobarbital ({approx}50 mg/kg IP). Under artificial ventilation, the aorta was promptly cannulated and the heart quickly excised. After retrograde coronary perfusion with normal Tyrode's solution at 36°C, nominally Ca2+-free Tyrode's solution was applied until contractions ceased. Next, Tyrode's solution containing 0.036 mmol/L Ca2+ and 0.4 mg/mL collagenase (type 1; Sigma Chemical Co) was retrogradely perfused for 20 minutes. Finally, the heart was perfused with KB solution33 at room temperature ({approx}21°C) to rinse away the collagenase. The partially digested heart was gently minced with scissors in KB solution. After filtering through a stainless steel mesh, cells were stored in the KB solution.

Electrophysiology
Pipettes were fabricated from borosilicate glass capillaries (Hilgenberg) by use of a two-step puller (model PP-83, Narishige). Pipettes had a tip resistance of 2 to 3 M{Omega} when filled with a 21 mmol/L Cl- pipette solution. Whole-cell currents were recorded at {approx}36°C by use of a patch-clamp amplifier (model EPC-7, List). Liquid junction potentials (-10 mV) were corrected by use of the voltage offset. Recorded currents were displayed directly on a chart recorder (Graphtec, Linearcorder WR 3320) and were fed to a computer (model PC-9801RA, NEC) at a sampling frequency of 0.5 kHz for on-line analyses with backup via an analog/digital convertor (model PCM-501ES, Sony) on videotapes. The holding potential was set at 0 mV to inactivate Na+ and Ca2+ channels. Voltage ramp pulses (±100 mV; -100 mV/s) were applied to record I-V relationships.

Measurements of cAMP Formation in Whole Cells
Guinea pig ventricular myocytes enzymatically isolated as for electrophysiological experiments were incubated in 500 µL of Krebs-Ringer buffer gassed with 95% O2/5% CO2 for 3 minutes. The myocytes ({approx}15 000 cells) were incubated in 500 µL of the buffer as a control in the presence or absence of isoproterenol (500 nmol/L or 1 µmol/L) with or without A-II (1 µmol/L) at 37°C for 30 minutes. The reaction was stopped by adding 100 µL of 30% trichloroacetic acid. Thereafter, the cAMP content of the cells was determined by RIA (Yamasa Shoyu Co).

Solutions and Drugs
Normal Tyrode's solution contained (in mmol/L) 143 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.5 NaH2PO4, 5.5 glucose, and 5 HEPES/NaOH (pH 7.4). KB solution contained (in mmol/L) 70 L-glutamic acid, 25 KCl, 10 taurine, 10 KH2PO4, 0.5 EGTA, 11 glucose, and 10 HEPES/KOH (pH 7.3). The internal pipette solution contained (in mmol/L) aspartic acid 85, EGTA 10, tetraethylammonium chloride 20, Na2 creatine phosphate 5, MgATP 10, MgCl2 0.5, glucose 5.5, and HEPES 10 and was titrated with CsOH (pH 7.4). Na2GTP (100 µmol/L) was added to the pipette solution to reduce rundown of G protein–mediated responses.34 Krebs-Ringer buffer contained (in mmol/L) 130 NaCl, 5.2 KCl, 2.8 CaCl2, 1.3 KH2PO4, 1.3 MgSO4, 24.8 NaHCO3, 3.3 glucose, 0.1 IBMX, 10 mg/mL BSA, 1 mg/mL bacitracin, and 10 HEPES/NaOH (pH 7.4). The Ca2+- and K+-free EPS contained (in mmol/L) 150 NaCl, 0.5 MgCl2, 5.5 glucose, 1 CdCl2, and 5 HEPES/NaOH (pH 7.4). When necessary, the Cl- concentration of the external solution was reduced to 20 mmol/L by replacing Cl- with equimolar L-aspartic acid.

PTX (Seikagaku Kougyou) was dissolved in KB solution (50 µg/mL stock solution) and was diluted to a final concentration of 5 µg/mL on addition to the myocyte suspension. The incubation was made at 36°C for >60 minutes. A-II was obtained from the Peptide Institute, Inc, isoproterenol from Nacalai Tesque, CV-11974 from the Takeda Chemical Industry, and PD123319 from Parke-Davis, Warner-Lambert Co. All other reagents were purchased from Sigma Chemical Co and Wako Industries, Ltd. All numerical data are expressed as mean±SEM (number of observations).


*    Results
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Exposure of a guinea pig ventricular myocyte to isoproterenol (40 nmol/L) produced outwardly rectifying currents (Fig 1A and 1BDownDown), which was attributed to activation of Cl- conductance through PKA.27 28 29 30 31 32 The isoproterenol-sensitive component was calculated as the difference of the I-V relations in the presence of agonist (b) and the baseline I-V relation (a). The reversal potential for the difference conductance (b-a in Fig 1CDown) was -47 mV, close to the estimated equilibrium potential for the 21-mmol/L Cl- pipette solution and 153-mmol/L Cl- external solution (-53 mV). Subsequent exposure to A-II (100 pmol/L and 1 nmol/L) reduced the Cl- current in a concentration-dependent manner; 100 pmol/L A-II reduced the amplitude (Fig 1CDown, c-a) by {approx}20%, and 1 nmol/L A-II reduced it by {approx}90% (Fig 1CDown, d-a). Reversal potentials for A-II–sensitive conductance were not affected by A-II, indicating that the peptide specifically reduces the membrane Cl- conductance.



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  		Figure 1. A-II inhibits isoproterenol-induced Cl- conductance in a concentration-dependent manner. A, Slow chart record of the whole-cell current at 0-mV holding potential. Arrow to the left of the chart indicates the 0 current level, and horizontal bars above the graph indicate extracellular application of isoproterenol and A-II. Arrows labeled a, b, c, and d correspond to those in B. B, I-V relations measured by ramp pulses as in A. a indicates the resting current; b, c, and d, after exposure to 40 nmol/L isoproterenol, 100 pmol/L A-II, and 1 nmol/L A-II, respectively. C, Difference currents showing the isoproterenol-induced current (b-a) and the current after application of 100 pmol/L (c-a) and 1 nmol/L (d-a) A-II. D, A-II concentration dependence for the inhibition of the 40-nmol/L isoproterenol-induced current. Difference current levels at +100 mV in various concentrations of A-II were normalized by those obtained in the presence of isoproterenol alone and are plotted as a function of A-II concentrations. Points represent mean±SEM. Numbers in parentheses indicate the number of observations. The curve represents a fit to a Hill equation: Control Response=100-Bmax/{1+[IC50/(A-II)]n}, where Bmax indicates maximal percent inhibition by A-II and n indicates the Hill coefficient.

Fig 1DUp illustrates the A-II concentration dependence of the Cl- current obtained from pooled data of 21 myocytes. Amplitudes of the Cl- currents elicited by isoproterenol (40 nmol/L) were measured at +100 mV in both the absence and presence of varying concentrations of A-II (for example, as b-a and c-a) and were normalized in reference to the amplitude induced by 40 nmol/L isoproterenol [for example, as 100x(c-a)/(b-a)]. The isoproterenol-elicited Cl- conductance could not be totally inhibited even by higher concentrations of A-II, and {approx}15% of the isoproterenol-induced conductance remained. Fitting to a logistic Hill equation (as described in the legend for Fig 1Up) yielded a half-maximal A-II concentration (IC50) of 0.24±0.08 nmol/L and a Hill coefficient (n) of 1.7±0.5.

To test the A-II receptor selectivity, we then used CV-11974, an AT1 receptor selective antagonist35 (Fig 2ADown), and PD123319, an AT2 receptor selective antagonist36 (Fig 2BDown). CV-11974 (1 µmol/L) completely prevented the inhibitory action of A-II on the ß-adrenergic activation of the Cl- current (n=3). On the other hand, PD123319 (100 nmol/L) failed to prevent this effect of A-II (percent inhibition was 83.8±6.9%, n=4). Therefore, AT1 receptors may mediate the inhibitory action of A-II.



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  		Figure 2. Sensitivity of the response to A-II receptor–selective compounds. In both A and B, chart records are shown on the left and difference currents on the right. Slow chart record of the whole-cell current at 0-mV holding potential. Arrows to the left of the charts indicate the 0 current level. A, Abolition of inhibition by A-II by preincubation with 1 µmol/L CV-11974. Chart record: Bars indicate exposure to 1 µmol/L CV-11974, 40 nmol/L isoproterenol, and 10 nmol/L A-II. Difference currents as indicated in the chart record show the effect of resting current (b-a); isoproterenol (c-a); A-II–sensitive (d-a) current in the presence of CV-11974; and that after washout of CV-11974 (e-a). B, PD123319 failed to prevent the inhibitory effect of A-II. Bars indicate exposure to 100 nmol/L PD123319 plus 50 nmol/L isoproterenol and 10 nmol/L A-II. Difference currents as indicated in the chart record show the isoproterenol-induced current (b-a) and A-II–sensitive current (c-a) in the presence of PD123319.

A muscarinic agonist, carbachol (1 µmol/L), decreased the isoproterenol-induced (40 nmol/L) conductance to 31.6±3.8% (n=4) without altering the reversal potential, presumably via inhibitory G proteins (Gi), which is in good agreement with previous reports.31 32 34 In the prolonged presence of carbachol, subsequent exposure of the cell to A-II (10 nmol/L) produced no additional inhibition (n=4; Fig 3ADown), suggesting that the intracellular signaling pathway may be common for carbachol and A-II.



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  		Figure 3. A-II antagonizes activation of adenylate cyclase. In both A and B, chart records are shown on the left and difference currents on the right. Slow chart record of the whole-cell current at 0-mV holding potential. Arrows to the left of the chart records indicate the 0 current level. A, Abolition of inhibition by A-II during the prolonged presence of 1 µmol/L carbachol (CCh). Chart record: Bars indicate exposure to 40 nmol/L isoproterenol, 1 µmol/L CCh, and 10 nmol/L A-II. Difference currents as indicated in the chart record show the isoproterenol-induced current (b-a), the current component after CCh (1 µmol/L) (c-a), and after CCh plus A-II (10 nmol/L) (d-a). B, Inhibition of the histamine-induced current. Chart record: Bars indicate exposure to 20 µmol/L histamine and 1 and 10 nmol/L A-II. Difference currents as indicated in the chart record show the histamine-induced current (b-a) and currents after A-II (c-a, d-a).

Histamine is known to increase cAMP via the stimulatory G protein (Gs)/adenylate cyclase pathway.31 A-II suppressed Cl- conductance in a dose-dependent manner when induced by histamine (20 µmol/L), as shown in Fig 3BUp. A-II (10 nmol/L) inhibited histamine-elicited conductance to 23.6±13.4% (n=4). This finding suggests that A-II antagonizes activation of adenylate cyclase by histamine or ß-adrenergic receptors.

AT1 receptors have been reported to belong to the G protein–coupled receptor superfamily with seven transmembrane segments.37 38 The involvement of G proteins in A-II inhibition of Cl- conductance was tested by use of a nonhydrolyzable guanine nucleotide, GTP{gamma}S. The nucleotide analogue is known to produce irreversible activation of G proteins of any type in an indistinguishable manner.39 Indeed, as represented in Fig 4Down, after the cells were internally dialyzed with GTP{gamma}S (100 µmol/L), brief exposure to isoproterenol (50 nmol/L) produced a persistent and irreversible activation of Cl- conductance, as previously demonstrated31 (Fig 4ADown). In the continued presence of pipette GTP{gamma}S, A-II could then no longer inhibit Cl- conductance (Fig 4A and 4BDownDown). The slight and continuous decrease of conductance after washing out isoproterenol is probably due to rundown of Cl- conductance or the PKA system. Reducing the Cl- concentration of EPS to 20 mmol/L decreased the outward current at 0 mV holding potential with a comparable shift of reversal potential as Cl- electrodes (Fig 4A and 4CDownDown). The remaining and persistently activating conductance was therefore sensitive to Cl-. Similar results were obtained in three myocytes, and the amplitude of the Cl- current was 86.8±5.3% of the initial value at the end of A-II application. Lack of inhibitory action of A-II was analogous to that of muscarinic agonists on Cl- conductance elicited by GTP{gamma}S plus isoproterenol,31 suggesting that A-II signal transduction is also G protein dependent.



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  		Figure 4. G proteins mediate A-II action. A, Slow chart record of the whole-cell current at 0-mV holding potential. Arrow to the left of the chart indicates the 0 current level. Bar at the bottom of the trace indicates internal dialysis with 100 µmol/L GTP{gamma}S. Bars at the top of the trace indicate extracellular application of 50 nmol/L isoproterenol and 10 nmol/L A-II and change of the external Cl- concentration to 20 mmol/L. B, Difference currents show the isoproterenol-induced current (b-a), after washout of isoproterenol (c-a), and after addition of A-II (d-a). C, Dependency of I-V relationship on external Cl- ions in the presence of 150 mmol/L (b-a) and 20 mmol/L (e-a) external Cl- concentrations.

To test the hypothesis that A-II may share a downstream signal transduction pathway with the carbachol receptor, the sensitivity of the G protein coupled to the A-II receptor to PTX was examined downstream from individual receptors. PTX uncouples receptors and Gi by ADP ribosylation of G proteins40 and abolishes carbachol inhibition of Cl- conductance.31 34 Although both carbachol and A-II inhibited the isoproterenol-elicited current in control myocytes (Fig 5ADown; see also Fig 3AUp), their effects were virtually prevented in the myocytes after prior incubation with PTX (5 µg/mL at 36°C >60 minutes; Fig 5BDown). Percent inhibition induced by A-II was only 4.4±1.3% (n=4). Thus, it is suggested that A-II receptors couple to a PTX-sensitive G protein, thereby resulting in the reduction of cAMP concentration and Cl- conductance.



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  		Figure 5. PTX-sensitive G proteins are involved in A-II action. In A and B, chart records are shown on the left and difference currents on the right. Slow chart record of the whole-cell current at 0-mV holding potential. Arrows to the left of the chart records indicate the 0 current level. A, Control cell. Chart record: Bars indicate exposure to 50 nmol/L isoproterenol, 1 µmol/L carbachol (CCh), and 10 nmol/L A-II. Difference currents as indicated in the chart record show the isoproterenol-induced current (b-a) and the currents after CCh (c-a) and after A-II (d-a). B, PTX-treated cell. Chart record: Bars as in A. Difference currents as in A.

Forskolin, a diterpene plant alkaloid, has been shown to increase intracellular cAMP by directly stimulating adenylate cyclase (for review, see Reference 41) and to evoke the same type of Cl- conductance as ß-adrenoceptor agonists and histamine.28 30 31 The forskolin-induced (500 nmol/L) Cl- current was also inhibited by A-II (10 and 100 nmol/L; Fig 6ADown), although its inhibitory effect seemed to be weaker than that on isoproterenol-induced Cl- current. In five myocytes, A-II at 10 and 100 nmol/L produced 22.7±1.9% and 34.3±6.2% inhibition of the forskolin-induced conductance, respectively.



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  		Figure 6. A-II acts by reducing the intracellular cAMP concentration. Slow chart record of the whole-cell current at 0-mV holding potential. Arrows to the left of the charts indicate the 0 current level. A, Inhibition of the forskolin-induced current. Chart record on the left in A: Bars indicate exposure to 500 nmol/L forskolin and 10 nmol/L and 100 nmol/L A-II. Difference currents as indicated in the chart record show the forskolin-induced current (b-a) and the currents after 10 nmol/L (c-a) and 100 nmol/L A-II (d-a). B, Absence of the effect of A-II on the current induced by internal dialysis with cAMP. Chart record of the whole-cell current. Bar at the bottom of the trace indicates internal dialysis with 1 mmol/L cAMP immediately on whole-cell formation. Bar at the top of the trace indicates exposure to 10 nmol/L A-II. At the time indicated by the vertical arrow, current gain for the chart recorder was reduced. C, A-II reduces the isoproterenol-induced increase of the ventricular cAMP level. Coordinate axis of the histogram shows percent change from the control cAMP concentration after exposure to 500 nmol/L isoproterenol (ISO), 500 nmol/L isoproterenol plus 1 µmol/L A-II, 1 µmol/L isoproterenol, or 1 µmol/L isoproterenol plus 1 µmol/L A-II. All experiments were measured in the presence of 0.1 mmol/L IBMX. The myocytes were incubated at 37°C for 30 minutes in Krebs-Ringer buffer with the compounds as indicated. Data represent mean±SEM (n=4 each). *No significance; **P<.05, significant difference from the increase seen with isoproterenol alone (factorial ANOVA with Fisher's protected least significant difference post hoc test).

Several studies report that the interaction between ß-adrenergic and muscarinic effects occurs at steps prior to the cAMP-dependent process.30 31 Therefore, we tested whether A-II works somewhere upstream from the production of cAMP. Dialysis of myocytes with intrapipette cAMP (1 mmol/L) elicited a large outwardly rectifying conductance soon after formation of the whole-cell patch-clamp configuration27 30 31 34 (Fig 6BUp). Lowering the Cl- concentration of EPS showed that the current so activated was Cl- sensitive (not shown in the figures, but see Fig 4CUp in Reference 32). A-II, even at >10 nmol/L, failed to inhibit the Cl- current elicited by intracellular cAMP (percent inhibition was 5.2±2.4%, n=4), supporting the hypothesis that A-II acts by reducing intracellular cAMP concentration through the inhibition of adenylate cyclase.

Finally, we directly examined the linkage between the A-II receptor stimulation and cAMP production by measuring the cAMP content of isolated guinea pig ventricular cells with a RIA (Fig 6CUp). Responses of cAMP to various agents are expressed as percent change from the control cAMP concentration. All the measurements were conducted in the continued presence of IBMX (0.1 mmol/L) to prevent the degradation of cAMP through phosphodiesterase activity. Isoproterenol increased the cAMP content in a concentration-dependent manner. A-II (1 µmol/L) significantly suppressed the increase of cAMP that had been induced by 500 nmol/L isoproterenol, although its effect was not significant on the 1-µmol/L isoproterenol-induced increase.


*    Discussion
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 *Discussion
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The present study shows that A-II inhibits the PKA-dependent Cl- conductance activated through the stimulation of ß-adrenoceptors and histamine type 2 receptors, which is analogous to the actions of muscarinic agonists and adenosine,27 28 29 30 31 because the effect of A-II was prevented by pretreatment with PTX, and because A-II could not affect the Cl- conductance activated by GTP{gamma}S plus isoproterenol or cAMP. Moreover, we observed by RIA that A-II reduced the isoproterenol-dependent increase of cAMP content. These results indicate that A-II may inhibit adenylate cyclase via Gi proteins. Several agonists, including muscarinic agonists30 31 and adenosine (M.H., MD, PhD, and L.-H.X., MS, unpublished data, 1995), have been shown to negatively couple to the intracellular PKA system via PTX-sensitive G proteins. More recently, we reported that the same signal transduction pathway is involved in endothelin type A receptor–mediated inhibition of chloride conductance.32 Thus, in the heart, multiple receptor/G protein networks are involved in modulating the enzyme activity of adenylate cyclase: activation via ß-adrenoceptors and histamine type 2 receptors, and suppression via A-II, adenosine, endothelin type A, and muscarinic receptors. It is of physiological and clinical significance that all agonists except ß-adrenergic and muscarinic stimulants are tissue hormones.

All the agonists that negatively couple to adenylate cyclase, but not A-II, have been known to open a distinct form of atrial K channels in a membrane-delimited manner. However, in preliminary experiments, we found that the atrial K channels were unaffected by A-II (M. Horie, MD, PhD, and T. Washizuka, MD, PhD, unpublished data, 1995). It is therefore likely that multiple receptors in the cardiac cell membrane share the same intracellular signal transduction pathway, but in a very specialized manner, depending on the type of agonists or receptors. Additional experiments will be necessary to elucidate this complicated network.

A-II may interact with AT1 receptors, because CV-11974, an AT1-selective antagonist, but not PD123319, an AT2-selective antagonist, prevented the inhibitory action. Using a receptor-binding assay, Baker and Singer7 reported two A-II binding sites in guinea pig ventricular cell membrane with Kd of 0.43 nmol/L and 3.6 nmol/L. The former value was close to the IC50 for A-II inhibition of Cl- conductance in the present study (0.24 nmol/L; Fig 1DUp). More recently, Habuchi and coworkers42 demonstrated that basal L-type Ca2+ current (ICa,L) in rabbit sinoatrial node cells is inhibited in a concentration-dependent manner by a nanomolar range of A-II via a PKA mechanism (IC50{cong}4 nmol/L). Although the overall action of A-II seems to be similar, there are several differences between their results and ours. A-II potency was {approx}20 times higher for the inhibition of the Cl- current than ICa,L. A-II (100 nmol/L) produced only a small inhibition of ICa,L ({approx}10%) that had been enhanced by 2 µmol/L isoproterenol, but A-II (100 nmol/L) suppressed the 1-µmol/L isoproterenol-induced Cl- conductance by 91±5% (n=3). Differences in preparations and currents are responsible in part for these discrepancies in data.

A-II receptors in the heart have been shown to link to membrane phosphoinositide breakdown by phospholipase C, thereby generating inositol 1,4,5-triphosphate and diacylglycerol and coupling to PKC activation (for review, see Reference 18). In the present study, sensitivity to PTX per se does not eliminate the possible involvement of this pathway for A-II–dependent inhibition of Cl- conductance, because some G proteins coupling to this system are known to be PTX sensitive.43 It is unlikely, however, because activities of phospholipase C and PKC are highly Ca2+ sensitive, and under the present experimental conditions, intracellular Ca2+ was tightly chelated to a very low concentration (0.1 to 1 nmol/L) by the use of Ca2+-free EPS and EGTA (10 mmol/L) in the pipette solution. On the contrary, activation of PKC has been shown to activate (but not inhibit) the same type of Cl- channel currents.44

The RIA measurement of cAMP production in isolated ventricular myocytes provided further evidence that A-II receptors negatively link to adenylate cyclase (Fig 6CUp). A-II (1 µmol/L) significantly antagonized the increase of cAMP induced by 500 nmol/L isoproterenol, consistent with the previous observation of Anand-Srivastava,21 who used isolated sarcolemmal fractions of rat heart cells. Although we failed to find significant suppression by A-II of 1-µmol/L isoproterenol-enhanced cAMP production, this might have been because of the addition of 0.1 mmol/L IBMX, which causes the accumulation of cAMP in the presence of potent ß-adrenoceptor stimulation.

The action of A-II on forskolin-induced Cl- conductance was weaker than that on isoproterenol-elicited Cl- conductance (Figs 1 and 6AUpUp), and A-II was no longer inhibitory on 10-µmol/L forskolin-elicited Cl- conductance (n=3; data not shown). A muscarinic receptor agonist, carbachol, was also found to fail to inhibit the 10-µmol/L forskolin-induced Cl- conductance.45 A similar inability of muscarinic agonists to inhibit ICa,L enhanced by a supermaximal concentration of forskolin has been reported in the same preparation (guinea pig ventricular myocytes46 ) and amphibian ventricular cells.47 48 These findings suggested that A-II may also alter the efficacy of forskolin but not its potency.

Such a discrepancy between adenylate cyclase activators may partially result from the type of enzyme that is actually activated: forskolin directly stimulates both membrane-bound and soluble cytosolic forms of enzyme. In contrast, isoproterenol specifically activates membrane-bound adenylate cyclase via Gs. In a receptor-mediated manner, A-II suppresses the membrane-bound enzyme but does not effectively suppress the soluble cytosolic one. A-II, therefore, might be a weak inhibitor of forskolin-induced conductance.

It has been demonstrated previously that PKA-dependent Cl- conductance is due to cardiac expression of the CFTR Cl- channel, an isoform of the epithelial CFTR gene product expressed because of alternative splicing.49 50 51 The existence of mRNA homologous to human CFTR has been shown in hearts of guinea pigs,49 rabbits,50 and humans.52 In humans, therefore, this type of Cl- conductance may affect cardiac function.

Plasma A-II concentrations range from 5 to 55 ng/L (5 to 55 pmol/L),53 54 and the tissue A-II level could be increased at very localized sites, especially under the pathological condition, in which sympathetic control has more substantial influence on the heart. A-II, therefore, may act as an autocrine and/or paracrine hormone, thereby directly modulating cardiac responses to catecholamines. More recently, we demonstrated55 that micromolar glibenclamide, an antidiabetic sulfonylurea agent, inhibited this type of Cl- channel and reversed the forskolin-induced shortening of action potential duration. Therefore, inhibition of Cl- conductance by A-II counteracts the excess shortening of action potential duration due to the increased catecholamines. This facilitates a larger transmembrane Ca2+ influx through ICa,L, thereby producing intracellular Ca overload. In addition, A-II–dependent inhibition of cAMP-stimulated Ca sequestration56 may also promote intracellular Ca overload. Such a sustained increase of intracellular Ca may account in part for A-II–mediated positive inotropic action9 10 11 and cellular hypertrophy after acute myocardial infarction12 13 14 and in animal models.15 16 Examination of the effects of A-II on Ca homeostasis by single-cell fluorometry will be the subject of future investigations.


*    Selected Abbreviations and Acronyms
 
A-II = angiotensin II
AT1, AT2 = angiotensin type 1, angiotensin type 2
CFTR = cystic fibrosis transmembrane conductance regulator
EPS = extracellular perfusion solution
IBMX = 3-isobutyl-1-methylxanthine
I-V = current-voltage
PKA = protein kinase A
PKC = protein kinase C
PTX = pertussis toxin
RIA = radioimmunoassay


*    Acknowledgments
 
The authors thank Dr A.F. James (King's College, London) for comments on the manuscript and Dr H. Matsubara for the kind gift of PD123319. This work was supported in part by grants-in-aid on priority areas of "Channel-Transporter Correlation" from the Japan Ministry of Education, Science and Culture. Dr Xie is a recipient of a scholarship from the Japan Ministry of Education, Science and Culture.

Received March 5, 1996; revision received August 20, 1996; accepted August 22, 1996.


*    References
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