Relationship between the increase in Ca²⁺ transient and contractile force induced by angiotensin II in aequorin-loaded rabbit ventricular myocardium

Abstract

Objectives: Pieces of evidence have been accumulating that imply a crucial role of angiotensin II (Ang II) in initiation and progress of heart failure, but the signalling processes subsequent to Ang II receptor activation in cardiac myocytes are complex and still controversial. We examined the effects of Ang II on the relationship between the intracellular Ca²⁺ transient and isometric contraction in mammalian ventricular myocardium. Methods: Isolated rabbit ventricular papillary muscle was loaded with the Ca²⁺ sensitive bioluminescent protein aequorin and electrically stimulated at a rate of 0.5 Hz at 37°C. Results: Ang II (10⁻⁸–10⁻⁶ M), in the presence of bupranolol (3×10⁻⁷ M) and prazosin (10⁻⁷ M), elicited a positive inotropic effect (PIE) in association with an increase in the peak Ca²⁺ transient. The maximal PIE of Ang II was about 30% of the isoproterenol-induced maximum (ISO_max), while the maximal increase in the peak Ca²⁺ transient induced by Ang II was only 7% of ISO_max. Ang II tended to prolong the duration of contraction (both time to peak force and relaxation time) but did not produce a discernible change in the duration of Ca²⁺ transient. The relationship between the amplitude of Ca²⁺ transient and peak force was shifted to the left by Ang II, as compared with the relationship for elevation of [Ca²⁺]_o (2.5–15.0 mM). The PIE and the increase in the amplitude of Ca²⁺ transient induced by Ang II were abolished by a selective angiotensin type 1 (AT₁) receptor antagonist losartan (10⁻⁵ M) but were not affected by a selective AT₂ receptor antagonist PD123319 (10⁻⁶ M). Conclusions: These results indicate that Ang II elicits a PIE through a dual mechanism via activation of AT₁ receptors in rabbit ventricular myocardium: by an increase in the amplitude of Ca²⁺ transient; and in addition by an increase in the myofilament Ca²⁺ sensitivity.

Angiotensin II, Myocardial contractility, Intracellular Ca²⁺ transient, Myofilament Ca²⁺ sensitivity, Losartan, AT₁ receptor antagonist, PD123319, AT₂ receptor antagonist

Ang II=angiotensin II, RAS=renin-angiotensin system, PIE=positive inotropic effect, AT₁=angiotensin type 1, AT₂=angiotensin type 2, ISO=isoproterenol, PI=phosphoinositide, IP₃=inositol 1,4,5-trisphosphate, PKC=protein kinase C, [Ca²⁺]_i=intracellular Ca²⁺ concentration, [Ca²⁺]_o=extracellular Ca²⁺ concentration, KHS=Krebs-Henseleit solution, EDTA=ethylenediaminetetraacetic acid, HEPES=N-2-hydroxyethylpiperazine-N″-2-ethanesulfonic acid, E-C=excitation-contraction, EIPA=ethylisopropyl amirolide

Time for primary review 36 days.

1 Introduction

Angiotensin II (Ang II) plays an important role in the regulation of haemodynamic homeostasis as well as cardiac hypertrophy and vascular remodeling in cardiovascular disorders such as congestive heart failure and myocardial ischemia [1, 2]. While the regulation by Ang II had been considered to be achieved through activation of renal renin-angiotensin system (RAS), it has recently become evident that local RAS in myocardial and vascular cells plays a crucial role in generation of Ang II and that Ang II is involved in the cardiovascular pathophysiological regulation in a manner much wider than has been postulated previously [3, 4]. Supporting the role of Ang II in pathophysiological regulation, the inhibition of RAS by angiotensin-converting enzyme (ACE) inhibitors or by angiotensin type 1 (AT₁) receptor antagonists has been revealed to be favorable for the treatment of these cardiovascular diseases, in which Ang II may play a crucial role in the progress of disorders [5–7].

While the activation of Ang II receptors is coupled to divergent intracellular signal transduction processes, the regulation by Ang II is in part mediated by an increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in various types of cell. Previous studies in vascular smooth muscle preparations have revealed [8, 9]that Ang II accelerates phosphoinositide (PI) hydrolysis to result in products acting as intracellular second messengers: inositol 1,4,5-trisphosphate (IP₃) that causes Ca²⁺ release from intracellular Ca²⁺ stores and thereby leads to an increase in [Ca²⁺]_i; and diacylglycerol that activates protein kinase C (PKC).

The effects of Ang II on [Ca²⁺]_i in myocardial cells, however, are rather controversial partly because of a wide range of species-dependent variation of contractile regulation among mammalian cardiac muscle. Ang II elicits a positive inotropic effect (PIE) in mammalian ventricular myocardium including rabbit [10–13]and human [14], and in chick heart [15]. But Ang II does not have any inotropic effect in multicellular ventricular muscle preparations of the dog and ferret [11]. In adult rat ventricular tissue, Ang II administration has been shown to elicit either a negative [16]or a positive [17]inotropic response. This bimodal response of rat ventricular tissue to Ang II may be partly due to differences in pre-contraction muscle length employed in these experiments [18]. In rabbit ventricular myocardium Ang II induces a PIE in association with an acceleration of PI hydrolysis [12].

The effects of Ang II on the intracellular Ca²⁺ transient in myocardial cells have also been rather controversial, which implies that a number of factors including the species of experimental animals employed, developmental changes in signal transduction processes, and the experimental procedures and conditions affect prominently the findings. In fura-2-loaded neonatal rat cardiomyocytes a transient increase followed by a sustained decrease in the amplitude of Ca²⁺ transient has been reported [19]. In fura-2-loaded cultured chick cardiomyocytes, Ang II also induced an increase in the amplitude of Ca²⁺ transient in association with acceleration of PI hydrolysis [20]. In contrast to these findings, in indo-1-loaded rabbit ventricular myocytes it was reported that Ang II induced a PIE without an increase in the amplitude of Ca²⁺ transients [10]. More recently, a slight increase in Ca²⁺ transients in an occasional cell [21]or a definite increase in the amplitude of Ca²⁺ transients by Ang II has been reported in indo-1-loaded rabbit ventricular myocytes [22]. The purpose of the present study was therefore twofold: (1) the first objective was to determine, with the use of aequorin, whether the inotropic effect of Ang II is accompanied by an increase in the amplitude of Ca²⁺ transient in rabbit ventricular myocardium; and (2) the second objective was to determine whether a part or all of the inotropic effect of Ang II can be attributed to the sensitization of myofilaments to Ca²⁺ ions.

2 Materials and methods

2.1 Simultaneous determination of the Ca²⁺ transient and contraction

Hearts were rapidly excised from male Japanese White rabbits (2.0–2.5 kg; 12–14 weeks) anaesthetized with sodium pentobarbital (50 mg/kg i.v.). Free-running thin papillary muscle (approximately 1 mm in diameter) was dissected from the right ventricle. Both ends of the muscle were tied with silk threads. Krebs-Henseleit solution (KHS), bubbled with 95% O₂–5% CO₂ and maintained pH 7.4, was used as the perfusate. The composition of KHS was as follows (mM): NaCl 118, KCl 4.7, CaCl₂ 2.5, MgCl₂ 1.2, NaHCO₃ 24.9, glucose 11.1 and ascorbic acid 0.057.

The Ca²⁺-sensitive bioluminescent protein aequorin was loaded by the modified chemical (macroinjection) technique as described previously [23]. The muscle was mounted horizontally in a 12 ml organ bath that contained low Ca²⁺ KHS (0.1 mM CaCl₂, 0.1 mM EDTA) at 25°C and exposed for 3–4 min. It is speculated that the low [Ca²⁺] in the loading solution may modify the cell membrane transiently and reversibly and allow intracellular entry of aequorin molecules surrounding the cells [24]. Aequorin was dissolved at a concentration of about 2 mg/ml in a solution containing 150 mM KCl and 5 mM HEPES buffer with a pH of 7.5. About 2–3 μl of aequorin were gently injected just beneath the endocardium through a fine-tipped glass micropipette. After aequorin had been injected, the concentration of CaCl₂ was gradually raised (0.2, 0.4, 0.8, 1.25 and 2.5 mM) at intervals of 15 min. Finally, low Ca²⁺ KHS was replaced with standard KHS and the temperature of KHS was raised to 37°C.

The aequorin-loaded muscle was transferred to a 50 ml organ bath specially designed for the simultaneous detection of aequorin luminescence and isometric tension. Experiments were carried out at 37°C. The muscle was electrically stimulated by square-wave pulses of 5 ms duration and a voltage about 10–20% above the threshold at 0.5 Hz (SEN 3201, Nihon Kohden, Tokyo, Japan). The aequorin luminescence was detected as current (nA) with a photomultiplier (9789A, Thorn EMI Electron Tubes, Ruislip, UK) and smoothed through a low-pass filter at 15 Hz. The isometric tension was detected with a strain-gauge transducer (Shinkoh UL-10 GR, Minebea, Tokyo, Japan). Both signals were recorded on a digital audio tape (PC-108M, Sony Magnescale, Tokyo, Japan) for later analysis. The length of the muscle was adjusted to give 90% of the maximal force [25], which may correspond to the sarcomere length of approximately ≤2.0 μm [26]. Preparations in which the resting tension increased progressively during an equilibration period were discarded. During the equilibration period of about 120 min, aequorin light declined to a steady low level, indicating that aequorin molecules that was present in the extracellular space had mostly been either washed out of the tissue during perfusion or remained in the interstitial space to be consumed (oxidized) rapidly when the Ca²⁺ concentration in the perfusate was increased to 1 mM [24].

Administration of Ang II and elevation of the extracellular Ca²⁺ concentration ([Ca²⁺]_o) were carried out in a cumulative manner by stepwise increases in concentration. When the increase in contractile force reached a steady level, the next higher concentration of the drug was administered. Prazosin (10⁻⁷ M), an α-adrenoceptor antagonist, and (±)-bupranolol (3×10⁻⁷ M), a β-adrenoceptor antagonist, were allowed to act for 30 min before and during administration of Ang II or elevation of [Ca²⁺]_o to avoid any possible modulation of the drug-induced response by endogenous norepinephrine that might be released during experiments. Finally, high concentrations of (−)-isoproterenol (ISO) were administered after washout of the drugs used. Preparations accepted for this study satisfied the following criteria: (1) aequorin light and contraction were stable during the course of experiments for several hours; and (2) the responsiveness of aequorin light and contraction to high concentrations of ISO remained high at the end of experiments.

Dimensions and mechanical parameters of papillary muscles used in the experiments were as follows (n=5): the average weight, 3.5±0.5 mg; length, 5.3±0.2 mm; cross-sectional area, 0.6±0.1 mm²; +dF/dt_max, 79.6±27.8 mN/s; −dF/dt_max, −87.5±29.4 mN/s.

One hundred signals of both aequorin light and contraction were averaged with a computer (ATAC-450, Nihon Kohden, Tokyo, Japan) to improve the signal-to-noise ratio. The peak of both signals was measured. The 2.5th root of the amplitude of aequorin light was calculated as an indicator of peak [Ca²⁺]_i, because the strength of aequorin luminescence varies approximately in proportion to the 2.5th power of the Ca²⁺ concentration [27]. The response of both signals to drugs was calculated as the value achieved by drugs minus the baseline value. The effect of drugs on the amplitude of both signals was expressed as a percentage of ISO_max (the maximal value induced by high concentrations of ISO minus the value after washout of the drug used) or as a percentage of the baseline value.

The possibility that Ang II, prazosin, losartan (2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2′-(1H-tetrazole-5-yl)biphenyl-4-yl)methyl]imidazole, potassium salt), PD123319 ((S)-1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid, ditrifluoroacetate, dihydrate) or CV-11974 (2-ethoxy-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-benzimidazole-7-carboxylic acid) might have direct effects on aequorin luminescence was examined by use of an in vitro detection apparatus [27]. A small volume of aequorin solution was rapidly injected into standard KHS in the absence or presence of these drugs. The amplitude and duration of aequorin luminescence were not changed in the presence of these drugs under the present experimental conditions (data not shown). Other drugs used in the experiments have previously been shown to have no direct effects on aequorin luminescence [23].

2.2 Statistical analysis

Experimental values are presented as means±S.E. The significance of differences between mean values was estimated by Student's t test. A value of P<0.05 was considered to indicate a significant difference.

2.3 Chemicals

The following drugs were used in the present experiments: Ang II (Peptide Institute, Osaka, Japan), losartan (Du Pont, Wilmington, DE, USA), PD123319 (Warner-Lambert, Ann Arbor, MI, USA), CV-11974 (Takeda Chemical Industries, Osaka, Japan), prazosin hydrochloride (Pfizer Taito, Tokyo, Japan), (±)-bupranolol hydrochloride (Kaken Pharmaceutical, Tokyo, Japan), (−)-isoproterenol hydrochloride (Sigma Chemical, St. Louis, MO, USA), sodium pentobarbital (Abbott Laboratories, North Chicago, IL, USA), and other chemicals (Wako Pure Chemical Industries, Osaka, Japan). Aequorin was purchased from Dr. J.R. Blinks (Friday Harbor Photoproteins, Friday Harbor, WA, USA).

3 Results

3.1 Effects of Ang II on the Ca²⁺ transient and contraction: comparison with effects of elevation of [Ca²⁺]_o

Ang II (10⁻⁸–10⁻⁶ M) elicited a concentration-dependent increase in the peak aequorin light and contractile force in the presence of prazosin (10⁻⁷ M) and (±)-bupranolol (3×10⁻⁷ M) as shown in Fig. 1 as the representative tracings during cumulative administration of Ang II. In the same preparation, a high concentration of ISO (10⁻⁵ M) elicited a prominent increase in the amplitude of aequorin light and contractile force.

Fig. 1

Representative tracings of the increases in isometric contractions (upper recordings) and in aequorin light transients (middle recordings) elicited by Ang II (10⁻⁸, 10⁻⁷, 10⁻⁶ M) and by ISO (10⁻⁵ M) in the same ventricular muscle excised from the rabbit. Lower recordings indicate stimulus signals. The response to Ang II was determined in the presence of prazosin (10⁻⁷ M) and (±)-bupranolol (3×10⁻⁷ M). The drugs used were washed out until the isometric contraction and aequorin light had returned to a level close to the control (not shown), and then ISO was administered. One hundred signals of both isometric contractions and aequorin light transients were averaged.

It has been reported in the rat ventricular muscle that Ang II has either a positive or a negative inotropic effect, depending on the muscle length [18]. The influence of the muscle length on the Ang II-induced PIE was, therefore, investigated in the rabbit papillary muscle unloaded with aequorin. By contrast to the rat, the PIE of Ang II was essentially the same at the length that gives 90% of the maximal force and at L_max (data not shown).

Fig. 2 shows the representative tracings during administration of Ang II (10⁻⁶ M) and elevation of [Ca²⁺]_o (10.0 mM) in the same preparation. Although Ang II (10⁻⁶ M) increased definitely the peak aequorin light, the PIE of Ang II was associated with much smaller increase in aequorin light as compared to when a similar PIE was caused by elevation of [Ca²⁺]_o (10.0 mM).

Fig. 2

Representative tracings of the increases in isometric contractions (upper recordings) and in aequorin light transients (middle recordings) during administration of Ang II (10⁻⁶ M) and elevation of [Ca²⁺]_o (10.0 mM) in the same ventricular muscle excised from the rabbit. The drugs used were washed out until the isometric contraction and aequorin light had returned to a level close to the control, and then [Ca²⁺]_o was increased. Lower recordings indicate stimulus signals. The responses to Ang II and elevation of [Ca²⁺] were determined in the presence of prazosin (10⁻⁷ M) and (±)-bupranolol (3×10⁻⁷ M). One hundred signals of both isometric contractions and aequorin light transients were averaged.

The duration of contraction tends to be slightly prolonged by Ang II (P>0.05) without a discernible change in the duration of aequorin light (Fig. 3). Elevation of [Ca²⁺]_o (10.0 mM) did not significantly affect the duration of contraction or aequorin light either (Fig. 3).

Fig. 3

Changes in duration of contraction (A) and of aequorin light (B) induced by Ang II (10⁻⁶ M) or elevation of [Ca²⁺]_o (10.0 mM). Values before administration of Ang II or elevation of [Ca²⁺]_o were taken as 100%. Data are means±S.E. (n=5). Values in the presence of Ang II were not significantly different from the respective control values. Actual values of duration of contraction and of aequorin light were as follows (in ms): 311.5±5.2 and 186.0±6.0 in total duration (open bars); 130.6±3.6 and 48.2±2.4 in time to peak (hatched bars); 180.9±5.5 and 137.8±6.2 in relaxation time (solid bars).

The concentrations-response curves for the increase in the amplitude of aequorin light and contractile force induced by Ang II (10⁻⁸–10⁻⁶ M) and elevation of [Ca²⁺]_o (2.5–15.0 mM) are shown in Fig. 4A and B. Ordinate is expressed in terms of the 2.5th root of the peak light based on the stoichiometry of light emission induced by Ca²⁺ bindings to aequorin molecules (for details, see Materials and Methods). Effects of Ang II and elevation of [Ca²⁺]_o on the (peak light)^1/2.5 and peak force were expressed as percentages of the respective baseline values. Ang II at 10⁻⁶ M produced a slight but significant increase in the (peak light)^1/2.5 (Fig. 4A). The maximal increase in the peak force was 335.8±49.7% of the baseline force, while the maximal increase in the (peak light)^1/2.5 was 152.2±17.0% of the baseline value (n=5, each). Elevation of [Ca²⁺]_o produced parallel increases in (peak light)^1/2.5 and peak force in a concentration-dependent manner (Fig. 4B). The maximal increase in the peak force induced by 15 mM [Ca²⁺]_o was 622.3±142.3% of the baseline force, and the maximal increase in the (peak light)^1/2.5 was 485.1±86.1% of the baseline value (n=5, each).

Fig. 4

A. Changes in the (peak light)^1/2.5 and peak force elicited by Ang II. Open and closed circles represent the changes in the (peak light)^1/2.5 and peak force elicited by Ang II (10⁻⁸, 10⁻⁷, 10⁻⁶ M) in the presence of prazosin (10⁻⁷ M) and (±)-bupranolol (3×10⁻⁷ M). Values before administration of Ang II were taken as 100%. Data are means±S.E. (n=5). *P<0.05 vs. the corresponding values before administration of Ang II. B. Changes in the (peak light)^1/2.5 and peak force elicited by elevation of [Ca²⁺]_o. Open and closed squares represent the changes in the (peak light)^1/2.5 and peak force elicited by elevation of [Ca²⁺]_o (5.0, 7.5, 10.0, 12.5, 15.0 mM) in the presence of prazosin (10⁻⁷ M) and (±)-bupranolol (3×10⁻⁷ M). Values before elevation of [Ca²⁺]_o were taken as 100%. Data are means±S.E. (n=5). *P<0.05 vs. the corresponding values before elevation of [Ca²⁺]_o.

The (peak light)^1/2.5-peak force relationship during administration of Ang II and elevation of [Ca²⁺]_o is shown in Fig. 5. The abscissa is expressed in terms of the 2.5th root of the peak aequorin light. Effects of Ang II and elevation of [Ca²⁺]_o on the (peak light)^1/2.5 and peak force were expressed as percentages of ISO_max. The maximal response of peak force to isoproterenol was 7.1±1.5 mN/mm², and ISO_max of peak light transients was 56.8±20.0 nA (n=5, each). The maximal increase in the peak force by Ang II was 27.7±5.8% of ISO_max, while the maximal increase in the (peak light)^1/2.5 induced by Ang II was only 6.7±2.4% of ISO_max (n=5, each). Elevation of [Ca²⁺]_o (2.5–15.0 mM) elicited a concentration-dependent PIE of 51.4±7.4% of ISO_max in association with an increase in the (peak light)^1/2.5 to 31.9±6.5% of ISO_max (n=5, each). The relationship during elevation of [Ca²⁺]_o was linear. While the relationship during administration of Ang II (10⁻⁸–10⁻⁶ M) was also linear, the relationship for Ang II was located to the left of that for elevation of [Ca²⁺]_o, indicating that Ang II elicits an increase in the myofibrillar Ca²⁺ sensitivity.

Fig. 5

The (peak light)^1/2.5-peak force relationship during administration of Ang II and elevation of [Ca²⁺]_o. The responses to Ang II and to elevation of [Ca²⁺]_o were determined in the presence of prazosin (10⁻⁷ M) and (±)-bupranolol (3×10⁻⁷ M). Open circles and squares represent the relationship during administration of Ang II (10⁻⁸, 10⁻⁷, 10⁻⁶ M) and elevation of [Ca²⁺]_o (5.0, 7.5, 10.0, 12.5, 15.0 mM), respectively. The abscissa is expressed in terms of the 2.5th root of the peak light (for details, see text). The effects of Ang II and elevation of [Ca²⁺]_o on the peak of both signals were expressed as percentages of ISO_max (the maximal values induced by ISO at 10⁻⁵ M minus the values after washout of the drugs used). Data are means±S.E. (n=5).

3.2 Influence of the AT₁ and AT₂ receptor antagonists

It was examined whether the effects of Ang II were mediated by AT₁ or AT₂ receptor. In this series of experiments Ang II (10⁻⁶ M) was administered first, and an AT₂ receptor antagonist PD123319 (10⁻⁵ M) and an AT₁ receptor antagonist losartan (10⁻⁶ M) were added successively in the presence of Ang II when the PIE of Ang II reached a steady level [11]. The elevated level of contractile force in the presence of Ang II had been maintained stable for 30–40 min [11]before the selective antagonists for the Ang II receptor subtype were administered in the presence of Ang II. The increase in the amplitude of aequorin light and the PIE induced by Ang II (10⁻⁶ M) were not affected by PD123319 (10⁻⁵ M), but they were almost completely inhibited by losartan (10⁻⁶ M) as shown in Fig. 6. Similar results were obtained in other 4 preparations (in one preparation, losartan was administered in the absence of PD 123319, and in another preparation, an AT₁ receptor antagonist CV-11974 (10⁻⁷ M) instead of losartan was employed).

Fig. 6

Representative tracings of the changes in isometric contractions (upper recordings) and of aequorin light transients (middle recordings) before and after administration of Ang II (10⁻⁶ M), and the influence of PD123319 (10⁻⁶ M) and losartan (10⁻⁵ M) added in the presence of Ang II in the same ventricular muscle excised from the rabbit. Lower recordings indicate stimulus signals. One hundred signals of both isometric contractions and aequorin light transients were averaged.

4 Discussion

In aequorin-loaded rabbit ventricular muscles Ang II at 10⁻⁸–10⁻⁶ M produced consistently a concentration-dependent increase in the peak Ca²⁺ transient (Figs. 1 and 2 and 4A), which was more pronounced than that observed in indo-1-loaded rabbit ventricular cardiomyocytes [21, 22]. While Ang II at 10⁻⁶ M produced a definite increase in the amplitude of Ca²⁺ transient, for an equivalent increase in contractile force, the increase in the amplitude of Ca²⁺ transient induced by Ang II was much smaller than that produced by an elevation of [Ca²⁺]_o (10 mM) (Fig. 2). In addition, in the presence of Ang II (10⁻⁸–10⁻⁶ M), the peak Ca²⁺ transient-peak force relationship was shifted to the left as compared with the relationship for an elevation of [Ca²⁺]_o (2.5–15.0 mM) (Fig. 5). These observations together with previous findings in indo-1-loaded single cardiomyocytes [10, 13, 21, 22]indicate that Ang II exerts an increase in the myofibrillar Ca²⁺ sensitivity in addition to an increase in the amplitude of Ca²⁺ transient.

Aequorin has been widely and successfully used as an indicator of [Ca²⁺]_i in a number of biological preparations including cardiac and vascular smooth muscle [28, 29]. Aequorin has certain limitations that are related to the insufficient sensitivity of aequorin molecule to low [Ca²⁺]: aequorin is not an ideal tool to measure diastolic [Ca²⁺]_i level in myocardial cells [27]but the aequorin light transient provides a good indication of the intracellular Ca²⁺ transient in contracting myocardial cells [28]. It is possible therefore to analyze the relationship between the amplitude of Ca²⁺ transients and contractile force during twitch contraction in intact myocardial cells and thereby to determine whether the drug administered modulates the myofibrillar Ca²⁺ sensitivity [30]. As the [Ca²⁺]_i level changes dynamically during twitch contraction and therefore the equilibrium of the kinetic relationship between the [Ca²⁺]_i and force generation is not reached [31], the inotropic agents, such as β-adrenoceptor agonists that abbreviate the duration of Ca²⁺ transients, produce an apparent shift of the relationship to the direction of desensitization of myofilaments to Ca²⁺ ions [25]. On the other hand, the effects on the myofibrillar Ca²⁺ sensitivity of the agents, such as Ang II and α-adrenoceptor agonists that have little effect on the duration of Ca²⁺ transients, can be analyzed without such an interference with the apparent modulation [25]. Thus the useful information about the modulation of myofibrillar Ca²⁺ sensitivity induced by inotropic agents is obtained by analyzing carefully the alterations in the amplitude and duration of both signals [23, 25, 29, 30]. In aequorin-loaded rabbit papillary muscle, elevation of [Ca²⁺]_o elicits a PIE in association with little change in duration of Ca²⁺ transients (Fig. 3) and is considered to be an intervention with least alteration of the myofibrillar Ca²⁺ sensitivity among available inotropic interventions [22, 23, 25]. Thus the effect of Ang II was compared with that of elevation of [Ca²⁺]_o as a standard (Fig. 5).

Ang II produced a definite increase in the amplitude of Ca²⁺ transients (Figs. 1 and 2 and 4A). A question arises how Ang II increases the amplitude of Ca²⁺ transients in rabbit papillary muscle. In the previous study, we found that neither a Ca²⁺ channel antagonist verapamil nor a Na⁺/H⁺ exchange inhibitor ethylisopropylamiloride (EIPA) alone abolished the PIE of Ang II but only the combination of both agents abolished the PIE of Ang II, an indication that both L-type Ca²⁺ channels and Na⁺/H⁺ exchanger may contribute to the elevation of [Ca²⁺]_i in the rabbit ventricular myocardium [32]. It has been shown that Ang II stimulates L-type Ca²⁺ channels [33–36]and Na⁺/H⁺ exchanger [13]in cardiac tissue.

In the rabbit ventricular myocardium, Ang II as well as α-adrenoceptor agonists and endothelin that stimulate PI hydrolysis elicited a PIE that is accompanied by an increase in myofibrillar Ca²⁺ sensitivity and a negative lusitropic effect [22, 37–40]. Endothelin and α-adrenoceptor agonists have been postulated to elicit a PIE through stimulation of Na⁺/H⁺ exchange by PKC activation due to endogenously generated diacylglycerol and resultant intracellular alkalinization in rat ventricular myocytes [41, 42]. Ang II has also been shown to activate Na⁺/H⁺ exchanger [13]to lead to an increase in intracellular pH [10]in rabbit ventricular myocardium. Therefore it appears to be highly likely that the Ang II-induced increase in the myofibrillar Ca²⁺ sensitivity may be mainly due to intracellular alkalinization through activation of Na⁺/H⁺ exchange, which is opposite in the direction with respect to pH that intracellular acidosis induces a decrease in the myofibrillar Ca²⁺ sensitivity both by a decrease in the affinity of troponin C for Ca²⁺[43]and by a direct depressant action on the cross-bridge interaction [44].

There are three alkalinizing transporters in cardiac myocytes including Na⁺/H⁺ exchange, Na⁺-dependent HCO₃⁻/Cl⁻ exchange and Na⁺/HCO₃⁻ symport [45–48]. Na⁺/HCO₃⁻ symport is activated by Ang II in cardiac cells through a mechanism that is independent of PI hydrolysis via activation of AT₂ receptors [49]. However, since the PIE of Ang II was inhibited by a selective AT₁ receptor antagonist losartan but not by an AT₂ receptor antagonist PD123319 (Fig. 6), activation of Na⁺/HCO₃⁻ symport may not play a role in the PIE of Ang II. The observation that the Na⁺/H⁺ exchange inhibitor such as EIPA selectively antagonized the PIE of Ang II without affecting the β-adrenoceptor-mediated PIE supports a crucial role of Na⁺/H⁺ exchanger in the PIE of Ang II in the rabbit ventricular myocardium [10, 13, 32].

Acknowledgements

This work was supported in part by Grants-in-Aid (nos. 06274202 and 06274201) for Scientific Research on Priority Areas and by a Grant-in-Aid for Scientific Research (B) (no. 0645155) from the Ministry of Education, Science, Sports and Culture, Japan. We are grateful to Du Pont, Pharmaceuticals and Biotechnology R and D (Wilmington, DE) for a generous supply of the selective AT₁ receptor antagonist losartan, to Dr. Joan A. Keiser, Warner-Lambert (Ann Arbor, MI) for a generous supply of the selective AT₂ receptor antagonist PD123319, to Takeda Chemical Industries (Osaka, Japan) for a generous supply of the selective AT₁ receptor antagonist CV-11974, to Pfizer Taito (Tokyo, Japan) for providing prazosin hydrochloride, and to Kaken Pharmaceutical (Tokyo, Japan) for providing (±)-bupranolol hydrochloride. We are also grateful to Prof. H. Tomoike for continuous support and encouragement for A. Watanabe to carry out the present study.

Author notes

1

Present address: Department of Internal Medicine I, Yamagata University School of Medicine, Yamagata 990-9585, Japan.