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Angiotensin II as a stimulator of Na+-dependent Ca2+ efflux from freshly isolated adult rat cardiomyocytes
ELSEVIER
Neuroscience Letters 213 (1996) 95-98
T
Angiotensin II as a stimulator of Na+-dependent Ca 2+ efflux from freshly isolated adult rat cardiomyocytes Y a s u s h i F u k u t a a, M a s a n o r i Y o s h i z u m i a'*, T e t s u y a K i t a g a w a a, T a k a k i H o r i a, F u m i o C h i k u g o a, T o m o h i s a K a w a h i t o a, I t s u o K a t o h a, H i t o s h i H o u c h i h, M o t o o O k a h aDepartment of Cardiovascular Surgery, School of Medicine, University of Tokushima, 2-50-1 Kuramoto,Tokushima 770, Japan bDepartment of Pharmacology, School of Medicine, University of Tokushima,Tokushima 770, Japan
Received 20 March 1996; revised version received 7 Jane 1996; accepted 19 June 1996
Abstract
In cardiac tissues, angiotensin II causes inotropic and chronotropic effects on the heart. It is indicated that the mechanism of the inotropic effect of angiotensin II is attributed to an increase in cytosolic free calcium ([Ca2+]i) in cardiomyocytes. However, increased [Ca2+]i should be restored to a physiological level because cumulative elevation in [Ca2+]i leads to irreversible injury in cardiomyocytes. Whereas it is known that angiotensin II causes the increase in [Ca2+]i in cardiac cells, little is known about the mechanisms of decrease in [Ca2+]i in cardiomyocytes upon angiotensin II stimulation. In the present study, we examined the effect of angiotensin II on Ca2÷ efflux from freshly isolated adult rat cardiomyocytes. Angiotensin II stimulated the efflux of 45Ca2÷ from the cells in a concentrationdependent manner (10-7-10 -5 M). The 45Ca2+ efflux from the cells was inhibited by type 1 angiotensin II receptor inhibitor. The angiotensin II-stimulated 45Ca2÷ efflux was not affected by deprivation of the extracellular Ca2÷, but was dependent on the presence of extracellular Na ÷. These results indicate that angiotensin II stimulates extracellular Na+-dependent 45Ca2÷ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on the plasma membrane type 1 angiotensin II receptors which may couple to Na÷/Ca 2÷ exchange. Keywords: Angiotensin II; Ca2+ efflux; Na+/Ca2+ exchange; Cardiomyocyte; Losartan; TCV-116
Non-adrenergic and non-cholinergic innervations to the cardiac tissue have been identified in recent years. For example, purine nucleotides may control cardiac cellular signal transduction through purinergic receptors [4]. The physiological role of ATP as a co-transmitter with noradrenaline has also been identified [18]. Adenosine is known to be involved in ischemic preconditioning of the heart and the existence of adenosine receptors on the heart has been explored [5]. As a candidate as a novel neurotransmitter, the role of nitric oxide in ischemic heart was proposed [13]. Recently, the renin-angiotensin system in the heart has been investigated. Angiotensin II (Ang II) is known to act as a growth factor in the heart and causes both inotropic and chronotropic changes within the heart [1]. It is also proposed that the effect of Ang II on the heart is partially mediated by an interaction with the sympa* Corresponding author. Tel.: +81 886 337152; fax: +81 886 337152.
thetic nervous system [16]. The existence of type 1 Ang II receptor (AT1) and that of type 2 Ang II receptor (AT2) are clarified in the myocardium [ 19]. Activation of AT1 by Ang II causes a positive inotropic effect on the heart [9]. It is assumed that the intracellular mechanism of Ang IIinduced effect is attributed to phosphoinositide hydrolysis which causes the elevation of cytosolic free calcium ([Ca2+]i) [10]. Increase in [Ca2+]i is considered to be a trigger for positive inotropic effect of the heart. However, this increased [Ca2+]i should be restored to a resting level for response to subsequent stimulation. Therefore, we examined the effect of Ang II on Ca 2÷ efflux from freshly isolated adult rat cardiomyocytes. Moreover, we investigated the mechanism of Ca 2÷ efflux from the cells induced by Ang II. Isolation of adult rat cardiomyocytes was performed by the method of Mitra and Morad [14] with minor modifications. Male Sprague-Dawley rats weighing 300-350 g
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(96)12842-1
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Y. Fukuta et al. / Neuroscience Letters 213 (1996) 95-98
were used. The rats were anesthetized by inhalation of saturated diethylether gas and were intravenously administered with heparin (1000 U/kg). The hearts were excised and the aortas were cannulated in a Langendorff perfusion model. Then, the hearts were perfused with calcium free buffer medium (medium A, 135 mM NaC1, 5.6 mM KC1, 1.0 mM MgC12, 0.33 mM NaH2PO4, I0.0 mM HEPES, and 0.2% bovine serum albumin (BSA), adjusted with NaOH to pH 7.30) for 3 min. Thereafter, the perfusion medium was changed to a cell dispersing medium with medium A supplemented with 0.2% collagenase I and 0.04% of protease XIV (medium B) for 12 min. After the digestion, these enzymes were washed out with medium C (medium A containing 0.22 mM of CaC12) for 3 min. After completion of the perfusion, the hearts were removed from the column and placed in a cluster dish containing 25 ml of medium C, supplemented with 2% of BSA (medium D). Small pieces of tissue (3 x 3 nun) were cut from the ventricle and gently shaken in 25 ml of medium D, thus releasing the dispersed cells. Then, the cells were resuspended once in 25 ml of calcium containing medium to prevent Ca 2÷ paradox [20] (0.44 mM of CaCI2 with medium D, a stepwise increase in Ca 2+ concentration) and centrifuged at 300 rev./min for 30 s. Afterwards, the supernatant was discarded and the cells were resusupended with modified Krebs-Henseleit bicarbonate buffer solution (135 mM NaCI, 5.6 mM KC1, 1.2 mM MgSO4, 1.2 mM K H 2 P O 4 , 25 mM NaHCO3, 2.2 mM CaCI2, and 10 mM glucose, adjusted with NaOH to pH 7.40). Then, the cell suspension was centrifuged again and finally, the isolated cardiomyocytes were resuspended in 5 ml of Krebs-Henseleit solution. The counted intact, rod-shaped cell numbers using hemocytometer were about 0.5 x 106 cells/ml. For measurement of 45Ca2* efflux from the cells, the isolated adult rat cardiomyocytes were incubated with 5 ml of KrebsHenseleit solution containing 45CaC12 (4/~Ci/ml) for 1 h at •37°C. Then 1.5 ml volume of the cell suspension (about 0.75 × l 0 6 cells) was resuspended and centrifuged two times with 10 ml of Krebs-Henseleit solution and applied onto the superfusion column consisting of 20 #m pore-size filter. The cells were then superfused with Krebs-Henseleit solution for 15 min to remove unincorporated 45Ca2" (flow rate was about 1 ml/min). Afterwards, the effluent was collected 15 times for a period of every 30 s (approximately 1 mVmin) to determine the basal efflux level. Then, the cells were superfused with a reaction mixture with or without Ang II. The effluent was collected 15 times for a period of every 30 s to determine the agonist-stimulated 45Ca 2+ efflux levels. After Ang II stimulation, the cells in the column were collected and solubilized in 1 ml of 1% Triton X-100 solution to determine the residual 45Ca2+ in the cells. The effluents were supplemented with 10 ml of liquid scintillation fluid and the radioactivities were counted in an Aloka 703 liquid scintillation counter for a 2 min period. The total radioactivity of 45Ca2+ in the cells was determined as the sum of the radioactivity in each
fraction and the residual radioactivity, and this value was used to calculate the fractional release of Ca 2. from the cells in each period. To investigate the influence of Ang II receptor antagonist on Ca 2÷ efflux induced by Ang II, putative AT1 antagonists, losartan and TCV-116 were used [15,19]. These antagonists were applied onto the columns 5 min prior to Ang II stimulation. For the experiment of Ca 2+ deprivation from the medium, Ca2+-free Krebs-Henseleit solution was used. Na ÷deficient medium was prepared with sucrose instead of Na + for the experiment of Na ÷ deprivation from the medium. The Ca 2+- and Na÷-deficient medium was applied onto the columns 2.5 min prior to Ang II administration. 45CaC12 was obtained from Amersham Corp., Tokyo, Japan. Ang II was purchased from Peptide Institute Inc., Osaka, Japan. Losartan was donated by Banyu Pharmaceutical Co. Ltd., Tokyo, Japan. TCV-116 was donated by Takeda Chemical Industries Ltd., Osaka, Japan. All other chemicals used were commercial products of reagent grade. Fig. 1 shows the efflux of 45Ca2+ from freshly isolated adult rat cardiomyocytes induced by various concentrations of Ang II. The stimulatory effect of Ang II on 45Ca2+ efflux was dose-dependent at concentrations of 10 -7 to 10 -5 M. The efflux of 45Ca2* increased to a peak value within about 1 min after the addition of Ang II. The peak value with 10 -5 M Ang II was 2.2 + 0.11% of total 45Ca2+ within the cells. After the peak, the efflux level decreased rapidly within the next 5 min.
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Time ( rain ) Fig. 1. Effects of various concentrations of Ang I1 on 45Ca2+ et'flux from freshly isolated adult rat cardiomyocytes. Cells were preloaded with 45Ca2+ as described in the text. After the stabilization of basal effiux level, Ang II was added and the cells were superfused for the next 7.5 min. Data are means for three to six separate experiments. The maximal SE was 0.11%. All peak levels with Ang II stimulation were significantly greater than the control level (P < 0.01).
97
Y. Fukuta et al. / Neuroscience Letters 213 (1996) 95-98
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Time ( min ) Fig. 2. Effects of ATI antagonists on 45Caa+ efflux from freshly isolated adult rat cardiomyocytes. Cells were preloaded with 45Ca2+ as described in the text. The AT1 antagonists, losartan or TCV-116, were added 5 min prior to submaximal concentration of Ang 1I (10 -6 M) stimulation. Data are means for three to six separate experiments. The peak levels with inhibitors were significantly less than that with Ang II only (P < 0.01).
To determine whether Ang II specifically stimulates 45Ca2+ efflux from the cardiomyocytes, we examined the effects of Ang II antagonists on its efflux. Fig. 2 shows the effects of AT1 antagonists, losartan and TCV-116 on the submaximal 45Ca2* efflux from the cells stimulated by 10-6 M Ang II. Ang II-induced Ca 2÷ was almost completely inhibited by both losartan and TCV-1 16 at a concentration of 10 -5 M. These results suggest that Ang II-induced 45Ca2+ efflux was mediated through AT1. Next, we examined whether Ang II-induced 45Ca2+ efflux is dependent on the presence of extracellular cations such as Ca 2÷ and Na ÷. We carried out a series of experiments in the absence of extracellular Ca 2+ and Na ÷. As shown in Fig. 3, Ang H-induced 45Ca2+ efflux was not influenced by the absence of extracellular Ca 2÷. However, Na+-free medium which was completely replaced by sucrose significantly inhibited Ang H-induced 45Ca2+ efflux from the cells. From these results, it is assumed that the effect of Ang II in stimulating Ca 2+ efflux across the plasma membrane may be mediated by an extracellular Na÷-dependent mechanism in isolated adult rat cardiomyocytes, presumably through Na+/Ca 2÷ exchange mechanism. Ang II has an important physiological and pathophysiological role in the control of blood pressure and plasma volume homeostasis [ 1 1]. Ang II exerts its hormonal, paracrine, or autocrine effects on many cells throughout the body (cardiovascular, renal, cerebral) at specific receptor sites. In cardiac tissues, Ang II acts as a cell growth factor [1], or as a modulator of sympathetic innervation [16]. Moreover, Ang II causes inotropic and chronotropic effects on the heart [1]. It is indicated that the mechanism of the inotropic effect of Ang II is attributed to an increase
in [Ca2+]i in cardiomyocytes [9]. However, increased [CaZ+]i should be restored to a physiological level because cumulative elevation in [Ca2+]i leads to irreversible injury in cardiomyocytes. It has been reported that the decline in [Ca2+]i may be due to the mechanism of Na+/Ca 2+ exchange at plasma membrane and Ca 2+ uptake into the sarcoplasmic reticulum [ 12]. Whereas it is known that Ang II causes the increase in [Ca2+]i in cardiac cells [10], little is known about the mechanism of decrease in [Ca2+]i in cardiomyocytes upon Ang II stimulation. Considering the above findings, it is conceivable that Ang II may have some biological effects on cardiomyocytes. Therefore, we hypothesized that Ang II may be involved in the reduction of [Ca2÷]~ in cardiomyocytes. In the present study, we examined for the first time the mechanisms by which Ang II stimulates Ca 2÷ efflux from freshly isolated adult rat cardiomyocytes. Results shown in Fig. 1 revealed that Ang II causes a significant Ca 2+ efflux from cardiomyocytes in a concentration-dependent manner. As the existence of Ang II receptor in cardiomyocyte has been reported, Ang II may affect the receptors on the cells. These Ang II receptors could be subclassified into two major groups such as AT1 and AT2 [19]. Next, we examined the effects of specific AT1 antagonists, losartan and TCV-1 16 on the effiux of Ca 2÷ from the cardiomyocytes [19]. As shown in Fig. 2, Ang II-induced Ca 2÷ was almost completely inhibited by both losartan and TCV-116 at a concentration of 10 -s M. 2.5o ~ 2.0-
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Time ( min ) Fig. 3. Influences of Ca2+- and Na+-deficient medium on Aug II-induced 45Ca2+ efflux from freshly isolated adult rat cardiomyocytes. Cells were preloaded with 4SCa2+ as described in the text. After the stabilization of basal efflux level, the medium was changed to Ca 2+- or Na+-deficient medium 2.5 rain prior to Aug II administration. Then, Ang II (10 -5 M) was added, and the cells were supeffused for 7.5 min. Na+-deficient medium was prepared with sucrose instead of Na +. Data are means for three to six separate experiments. The maximal SE was 0.10%. The peak level with Ang II stimulation in Na+-deficient medium was significantly less than that with Aug II in normal medium (P < 0.01).
98
Y. Fukuta et al. / Neuroscience Letters 213 (1996) 95-98
From these results, it can be assumed that the stimulatory effect of Ang II on Ca 2+ efflux from cardiomyocytes is mediated by AT1. To clarify the mechanism ofAng II-induced Ca 2÷ efflux from the cells, we further examined the influence of deprivation of extracellular Ca 2+ or Na + from the medium. Ang II-induced Ca 2÷ efflux from cardiomyocytes was dependent on the presence of extracellular Na +, but not on Ca 2+ (Fig. 3). These results suggest that Ang II stimulated Ca 2÷ efflux from cardiomyocytes through the mechanism of Na+/Ca 2+ exchange. The deprivation of Na ÷ from the incubation medium represents the inhibition of Na+/Ca 2÷ exchange which will work to extrude intracellular Ca 2÷ outside the cells in turn of Na + influx. Thus, it is assumed that the acceleration of Na+/Ca 2÷ exchange on the plasma membrane has a key role in Ang II-induced Ca 2÷ efflux from cardiomyocytes. Activation of Ang II receptors in cardiomyocytes is coupled to various signal-transduction processes, which include stimulation of the slow Ca 2÷ channel [7] and acceleration of the hydrolysis of phosphoinositide [10], or activation of protein kinase C [6]. Moreover, it has also been reported that Ang II induces Ca 2÷ efflux from bovine adrenal chromaffin cells through Na+/Ca 2÷ exchange mechanism [8]. Considering these findings, it may be reasonable to speculate that Ang II-induced Ca 2÷ efflux from adult rat cardiomyocytes may be mediated by the mechanism of Na+/Ca 2+ exchange and which may accelerate Ca 2÷ efflux from the cardiomyocytes by the stimulation of inositol triphosphate turnover coupled with ATI receptors. These results are consistent with the report that Ang II stimulates Na+/Ca 2÷ exchanger in rat heart [2]. On the other hand, it has also been reported that inositoi 1,4,5-trisphosphate (IP3), which is produced by the stimulation of inositol triphosphate turnover, increases the [Ca2+]i in cardiomyocytes [3], Ang II may stimulate simultaneously the extrusion of this elevated [Ca2+]i to a resting level as a counter action for increase in [Ca2+]i within the cells. Smith et al. reported that Ang II evoked 4SCa2+ efflux from cultured arterial muscle cells concomitantly with the elevation of [Ca2+]i [17]. However, it is still unclear how Ang II causes the 45Ca 2+ efflux from the cardiomyocytes. We are now investigating the intracellular mechanism which is involved in Ang II-induced Ca 2÷ efflux from freshly isolated adult rat cardiomyocytes. In conclusion, Ang II stimulates Ca 2+ efftux from adult rat cardiomyocytes probably through AT1 receptor. The underlying mechanism, which causes Ca 2+ efflux from the cells is still not entirely understood, however it can be explained at least in part by Na+/Ca 2+ exchange on the plasma membrane. [1] Baker, K.M. and Aceto, J.F., Angiotensin II stimulation of protein synthesis and cell growth in chick heart cells, Am. J. Physiol., 259 (1990) H610-H618.
[2] Ballard, C. and Schaffer, S., Stimulation of the Na+/Ca2+ exchanger by phenylephrine, angiotensin II and endothelin I, J. Mol. Cell. Cardiol., 28 (1996) 11-17. [3] Berridge, M.J., Inositol trisphosphate and calcium signaling, Nature, 361 (1993) 315-325. [4] De Young, M.B. and Scarpa, A., Extracellular ATP induces Ca2+ transients in cardiac myocytes which are potentiated by norepinephrine, FEBS Lett., 223 (1987) 53-58. [5] Downey, LM., Liu, G.S. and Thornton, J.D., Adenosine and the anti-infarct effects of preconditioning, Cardiovasc. Res., 27 (1993) 3-8. [6] Duff, J.L., Marrero, M.B., Paxton, W.G., Schieffer, B., Bemstein, K.E. and Berk, B.C., Angiotensin II signal transduction and the mitogen-activated protein kinase pathway, Cardiovasc. Res., 30 (1995) 511-517. [7] Freer, R.J., Pappano, A.J., Peach, M.J., Bing, K.T., McLean, M.J., Vogel, S. and Sperelakis, N., Mechanism for the positive inotropic effect of angiotensin II on isolated cardiac muscle, Circ. Res., 39 (1976) 178-183. [8] Houchi, H., Okuno, M., Kitamura, K., Ishimura, Y., Ohuchi, T., Tokumura, A. and Oka, M., Stimulatory effect of angiotensin II on calcium efflux from cultured bovine adrenal chromaffin cells, Life Sci., 56 (1995) 109-114. [9] Ishihata, A. and Endoh, M., Species-related differences in inotropic effects of angiotensin II in mammalian ventricular muscle: receptors, subtypes and phosphoinositide hydrolysis, Br. J. Pharmacol., 114 (1995) 447-453. [10] Kem, D.C., Johnson, E.I.M., Capponi, A.M., Chardonnens, D., Lang, U., Blondel, B., Kosida, H. and Vallotton, M.B., Effect of angiotensin 11 on cytosolic free calcium in neonatal rat cardiomyocytes, Am. J. Physiol., 261 (1991) C77-C85. [I 1] Laragh, J.H., The lenin system and new understanding of the complications of hypertension and their treatment, Arzneim.-ForschJ Drug Res., 43 (1993) 247-254. [12] Lewartowski, B., Wolska, B.M. and Zdanowski, K., The effects of blocking the Na-Ca exchange at intervals throughout the physiological contraction-relaxation cycle of single cardiac myocyte, J. Mol. Cell. Cardiol., 24 (1992) 967-976. [13] Maulik, N., Engelman, D.T., Watanabe, M., Engelman, R.M., Maulik, G., Cordis, G.A. and Das, D.K., Nitric oxide signaling in ischemic heart, Cardiovasc. Res., 30 (1995) 593-601. [14] Mitra, R. and Morad, M., A uniform enzymatic method for dissociation of myocytes from hearts and stomachs of vertebrates, Am. J. Physiol., 249 (1985) H1056-H1060. [15] Ogihara, T., Higashimori, K., Masuo, K. and Mikami, H., Pilot study of a new angiotensin II receptor antagonist, TCV-116: effects of a single oral dose on blood pressure in patients with essential hypertension, Ciin. Therapeutics, 15 (1993) 684-691. [16] Sexena, P.R., Interaction between the renin-angioteasin-aldosterone and sympathetic nervous systems, J. Cardiovasc. Pharmacol., 19 (1992) $80-$88. [17] Smith, J.B. and Smith, L., Extracellular Na ÷ dependence of changes in free Ca2÷, 45Ca2+ efflux, and total cell Ca~+ produced by angiotensin 11 in cultured arterial muscle cells, J. Biol. Chem., 262 (1987) 17455-17460. [18] Sneddon, P. and Bumstock, G., ATP as a co-transmitter in rat tail artery, Ear. J. Pharmacol., 106 (1985) 149-152. [19] Timmermans, P.B.M.W.M., Wong, P.C., Chiu, A.T. and Herblin, W.F., Nonpeptide angiotensin 11receptor antagonists, Trends Pharmacol. Sci., 12 (1991) 55-62. [20] Zimmerman, A.N.E. and Hulsmann, W.C., Paradoxical influence of calcium ions on the permeability of the cell membranes of the isolated rat heart, Nature, 211 (1966) 646-647.
Neuroscience Letters 213 (1996) 95-98
T
Angiotensin II as a stimulator of Na+-dependent Ca 2+ efflux from freshly isolated adult rat cardiomyocytes Y a s u s h i F u k u t a a, M a s a n o r i Y o s h i z u m i a'*, T e t s u y a K i t a g a w a a, T a k a k i H o r i a, F u m i o C h i k u g o a, T o m o h i s a K a w a h i t o a, I t s u o K a t o h a, H i t o s h i H o u c h i h, M o t o o O k a h aDepartment of Cardiovascular Surgery, School of Medicine, University of Tokushima, 2-50-1 Kuramoto,Tokushima 770, Japan bDepartment of Pharmacology, School of Medicine, University of Tokushima,Tokushima 770, Japan
Received 20 March 1996; revised version received 7 Jane 1996; accepted 19 June 1996
Abstract
In cardiac tissues, angiotensin II causes inotropic and chronotropic effects on the heart. It is indicated that the mechanism of the inotropic effect of angiotensin II is attributed to an increase in cytosolic free calcium ([Ca2+]i) in cardiomyocytes. However, increased [Ca2+]i should be restored to a physiological level because cumulative elevation in [Ca2+]i leads to irreversible injury in cardiomyocytes. Whereas it is known that angiotensin II causes the increase in [Ca2+]i in cardiac cells, little is known about the mechanisms of decrease in [Ca2+]i in cardiomyocytes upon angiotensin II stimulation. In the present study, we examined the effect of angiotensin II on Ca2÷ efflux from freshly isolated adult rat cardiomyocytes. Angiotensin II stimulated the efflux of 45Ca2÷ from the cells in a concentrationdependent manner (10-7-10 -5 M). The 45Ca2+ efflux from the cells was inhibited by type 1 angiotensin II receptor inhibitor. The angiotensin II-stimulated 45Ca2÷ efflux was not affected by deprivation of the extracellular Ca2÷, but was dependent on the presence of extracellular Na ÷. These results indicate that angiotensin II stimulates extracellular Na+-dependent 45Ca2÷ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on the plasma membrane type 1 angiotensin II receptors which may couple to Na÷/Ca 2÷ exchange. Keywords: Angiotensin II; Ca2+ efflux; Na+/Ca2+ exchange; Cardiomyocyte; Losartan; TCV-116
Non-adrenergic and non-cholinergic innervations to the cardiac tissue have been identified in recent years. For example, purine nucleotides may control cardiac cellular signal transduction through purinergic receptors [4]. The physiological role of ATP as a co-transmitter with noradrenaline has also been identified [18]. Adenosine is known to be involved in ischemic preconditioning of the heart and the existence of adenosine receptors on the heart has been explored [5]. As a candidate as a novel neurotransmitter, the role of nitric oxide in ischemic heart was proposed [13]. Recently, the renin-angiotensin system in the heart has been investigated. Angiotensin II (Ang II) is known to act as a growth factor in the heart and causes both inotropic and chronotropic changes within the heart [1]. It is also proposed that the effect of Ang II on the heart is partially mediated by an interaction with the sympa* Corresponding author. Tel.: +81 886 337152; fax: +81 886 337152.
thetic nervous system [16]. The existence of type 1 Ang II receptor (AT1) and that of type 2 Ang II receptor (AT2) are clarified in the myocardium [ 19]. Activation of AT1 by Ang II causes a positive inotropic effect on the heart [9]. It is assumed that the intracellular mechanism of Ang IIinduced effect is attributed to phosphoinositide hydrolysis which causes the elevation of cytosolic free calcium ([Ca2+]i) [10]. Increase in [Ca2+]i is considered to be a trigger for positive inotropic effect of the heart. However, this increased [Ca2+]i should be restored to a resting level for response to subsequent stimulation. Therefore, we examined the effect of Ang II on Ca 2÷ efflux from freshly isolated adult rat cardiomyocytes. Moreover, we investigated the mechanism of Ca 2÷ efflux from the cells induced by Ang II. Isolation of adult rat cardiomyocytes was performed by the method of Mitra and Morad [14] with minor modifications. Male Sprague-Dawley rats weighing 300-350 g
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(96)12842-1
96
Y. Fukuta et al. / Neuroscience Letters 213 (1996) 95-98
were used. The rats were anesthetized by inhalation of saturated diethylether gas and were intravenously administered with heparin (1000 U/kg). The hearts were excised and the aortas were cannulated in a Langendorff perfusion model. Then, the hearts were perfused with calcium free buffer medium (medium A, 135 mM NaC1, 5.6 mM KC1, 1.0 mM MgC12, 0.33 mM NaH2PO4, I0.0 mM HEPES, and 0.2% bovine serum albumin (BSA), adjusted with NaOH to pH 7.30) for 3 min. Thereafter, the perfusion medium was changed to a cell dispersing medium with medium A supplemented with 0.2% collagenase I and 0.04% of protease XIV (medium B) for 12 min. After the digestion, these enzymes were washed out with medium C (medium A containing 0.22 mM of CaC12) for 3 min. After completion of the perfusion, the hearts were removed from the column and placed in a cluster dish containing 25 ml of medium C, supplemented with 2% of BSA (medium D). Small pieces of tissue (3 x 3 nun) were cut from the ventricle and gently shaken in 25 ml of medium D, thus releasing the dispersed cells. Then, the cells were resuspended once in 25 ml of calcium containing medium to prevent Ca 2÷ paradox [20] (0.44 mM of CaCI2 with medium D, a stepwise increase in Ca 2+ concentration) and centrifuged at 300 rev./min for 30 s. Afterwards, the supernatant was discarded and the cells were resusupended with modified Krebs-Henseleit bicarbonate buffer solution (135 mM NaCI, 5.6 mM KC1, 1.2 mM MgSO4, 1.2 mM K H 2 P O 4 , 25 mM NaHCO3, 2.2 mM CaCI2, and 10 mM glucose, adjusted with NaOH to pH 7.40). Then, the cell suspension was centrifuged again and finally, the isolated cardiomyocytes were resuspended in 5 ml of Krebs-Henseleit solution. The counted intact, rod-shaped cell numbers using hemocytometer were about 0.5 x 106 cells/ml. For measurement of 45Ca2* efflux from the cells, the isolated adult rat cardiomyocytes were incubated with 5 ml of KrebsHenseleit solution containing 45CaC12 (4/~Ci/ml) for 1 h at •37°C. Then 1.5 ml volume of the cell suspension (about 0.75 × l 0 6 cells) was resuspended and centrifuged two times with 10 ml of Krebs-Henseleit solution and applied onto the superfusion column consisting of 20 #m pore-size filter. The cells were then superfused with Krebs-Henseleit solution for 15 min to remove unincorporated 45Ca2" (flow rate was about 1 ml/min). Afterwards, the effluent was collected 15 times for a period of every 30 s (approximately 1 mVmin) to determine the basal efflux level. Then, the cells were superfused with a reaction mixture with or without Ang II. The effluent was collected 15 times for a period of every 30 s to determine the agonist-stimulated 45Ca 2+ efflux levels. After Ang II stimulation, the cells in the column were collected and solubilized in 1 ml of 1% Triton X-100 solution to determine the residual 45Ca2+ in the cells. The effluents were supplemented with 10 ml of liquid scintillation fluid and the radioactivities were counted in an Aloka 703 liquid scintillation counter for a 2 min period. The total radioactivity of 45Ca2+ in the cells was determined as the sum of the radioactivity in each
fraction and the residual radioactivity, and this value was used to calculate the fractional release of Ca 2. from the cells in each period. To investigate the influence of Ang II receptor antagonist on Ca 2÷ efflux induced by Ang II, putative AT1 antagonists, losartan and TCV-116 were used [15,19]. These antagonists were applied onto the columns 5 min prior to Ang II stimulation. For the experiment of Ca 2+ deprivation from the medium, Ca2+-free Krebs-Henseleit solution was used. Na ÷deficient medium was prepared with sucrose instead of Na + for the experiment of Na ÷ deprivation from the medium. The Ca 2+- and Na÷-deficient medium was applied onto the columns 2.5 min prior to Ang II administration. 45CaC12 was obtained from Amersham Corp., Tokyo, Japan. Ang II was purchased from Peptide Institute Inc., Osaka, Japan. Losartan was donated by Banyu Pharmaceutical Co. Ltd., Tokyo, Japan. TCV-116 was donated by Takeda Chemical Industries Ltd., Osaka, Japan. All other chemicals used were commercial products of reagent grade. Fig. 1 shows the efflux of 45Ca2+ from freshly isolated adult rat cardiomyocytes induced by various concentrations of Ang II. The stimulatory effect of Ang II on 45Ca2+ efflux was dose-dependent at concentrations of 10 -7 to 10 -5 M. The efflux of 45Ca2* increased to a peak value within about 1 min after the addition of Ang II. The peak value with 10 -5 M Ang II was 2.2 + 0.11% of total 45Ca2+ within the cells. After the peak, the efflux level decreased rapidly within the next 5 min.
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Time ( rain ) Fig. 1. Effects of various concentrations of Ang I1 on 45Ca2+ et'flux from freshly isolated adult rat cardiomyocytes. Cells were preloaded with 45Ca2+ as described in the text. After the stabilization of basal effiux level, Ang II was added and the cells were superfused for the next 7.5 min. Data are means for three to six separate experiments. The maximal SE was 0.11%. All peak levels with Ang II stimulation were significantly greater than the control level (P < 0.01).
97
Y. Fukuta et al. / Neuroscience Letters 213 (1996) 95-98
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Time ( min ) Fig. 2. Effects of ATI antagonists on 45Caa+ efflux from freshly isolated adult rat cardiomyocytes. Cells were preloaded with 45Ca2+ as described in the text. The AT1 antagonists, losartan or TCV-116, were added 5 min prior to submaximal concentration of Ang 1I (10 -6 M) stimulation. Data are means for three to six separate experiments. The peak levels with inhibitors were significantly less than that with Ang II only (P < 0.01).
To determine whether Ang II specifically stimulates 45Ca2+ efflux from the cardiomyocytes, we examined the effects of Ang II antagonists on its efflux. Fig. 2 shows the effects of AT1 antagonists, losartan and TCV-116 on the submaximal 45Ca2* efflux from the cells stimulated by 10-6 M Ang II. Ang II-induced Ca 2÷ was almost completely inhibited by both losartan and TCV-1 16 at a concentration of 10 -5 M. These results suggest that Ang II-induced 45Ca2+ efflux was mediated through AT1. Next, we examined whether Ang II-induced 45Ca2+ efflux is dependent on the presence of extracellular cations such as Ca 2÷ and Na ÷. We carried out a series of experiments in the absence of extracellular Ca 2+ and Na ÷. As shown in Fig. 3, Ang H-induced 45Ca2+ efflux was not influenced by the absence of extracellular Ca 2÷. However, Na+-free medium which was completely replaced by sucrose significantly inhibited Ang H-induced 45Ca2+ efflux from the cells. From these results, it is assumed that the effect of Ang II in stimulating Ca 2+ efflux across the plasma membrane may be mediated by an extracellular Na÷-dependent mechanism in isolated adult rat cardiomyocytes, presumably through Na+/Ca 2÷ exchange mechanism. Ang II has an important physiological and pathophysiological role in the control of blood pressure and plasma volume homeostasis [ 1 1]. Ang II exerts its hormonal, paracrine, or autocrine effects on many cells throughout the body (cardiovascular, renal, cerebral) at specific receptor sites. In cardiac tissues, Ang II acts as a cell growth factor [1], or as a modulator of sympathetic innervation [16]. Moreover, Ang II causes inotropic and chronotropic effects on the heart [1]. It is indicated that the mechanism of the inotropic effect of Ang II is attributed to an increase
in [Ca2+]i in cardiomyocytes [9]. However, increased [CaZ+]i should be restored to a physiological level because cumulative elevation in [Ca2+]i leads to irreversible injury in cardiomyocytes. It has been reported that the decline in [Ca2+]i may be due to the mechanism of Na+/Ca 2+ exchange at plasma membrane and Ca 2+ uptake into the sarcoplasmic reticulum [ 12]. Whereas it is known that Ang II causes the increase in [Ca2+]i in cardiac cells [10], little is known about the mechanism of decrease in [Ca2+]i in cardiomyocytes upon Ang II stimulation. Considering the above findings, it is conceivable that Ang II may have some biological effects on cardiomyocytes. Therefore, we hypothesized that Ang II may be involved in the reduction of [Ca2÷]~ in cardiomyocytes. In the present study, we examined for the first time the mechanisms by which Ang II stimulates Ca 2÷ efflux from freshly isolated adult rat cardiomyocytes. Results shown in Fig. 1 revealed that Ang II causes a significant Ca 2+ efflux from cardiomyocytes in a concentration-dependent manner. As the existence of Ang II receptor in cardiomyocyte has been reported, Ang II may affect the receptors on the cells. These Ang II receptors could be subclassified into two major groups such as AT1 and AT2 [19]. Next, we examined the effects of specific AT1 antagonists, losartan and TCV-1 16 on the effiux of Ca 2÷ from the cardiomyocytes [19]. As shown in Fig. 2, Ang II-induced Ca 2÷ was almost completely inhibited by both losartan and TCV-116 at a concentration of 10 -s M. 2.5o ~ 2.0-
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Time ( min ) Fig. 3. Influences of Ca2+- and Na+-deficient medium on Aug II-induced 45Ca2+ efflux from freshly isolated adult rat cardiomyocytes. Cells were preloaded with 4SCa2+ as described in the text. After the stabilization of basal efflux level, the medium was changed to Ca 2+- or Na+-deficient medium 2.5 rain prior to Aug II administration. Then, Ang II (10 -5 M) was added, and the cells were supeffused for 7.5 min. Na+-deficient medium was prepared with sucrose instead of Na +. Data are means for three to six separate experiments. The maximal SE was 0.10%. The peak level with Ang II stimulation in Na+-deficient medium was significantly less than that with Aug II in normal medium (P < 0.01).
98
Y. Fukuta et al. / Neuroscience Letters 213 (1996) 95-98
From these results, it can be assumed that the stimulatory effect of Ang II on Ca 2+ efflux from cardiomyocytes is mediated by AT1. To clarify the mechanism ofAng II-induced Ca 2÷ efflux from the cells, we further examined the influence of deprivation of extracellular Ca 2+ or Na + from the medium. Ang II-induced Ca 2÷ efflux from cardiomyocytes was dependent on the presence of extracellular Na +, but not on Ca 2+ (Fig. 3). These results suggest that Ang II stimulated Ca 2÷ efflux from cardiomyocytes through the mechanism of Na+/Ca 2+ exchange. The deprivation of Na ÷ from the incubation medium represents the inhibition of Na+/Ca 2÷ exchange which will work to extrude intracellular Ca 2÷ outside the cells in turn of Na + influx. Thus, it is assumed that the acceleration of Na+/Ca 2÷ exchange on the plasma membrane has a key role in Ang II-induced Ca 2÷ efflux from cardiomyocytes. Activation of Ang II receptors in cardiomyocytes is coupled to various signal-transduction processes, which include stimulation of the slow Ca 2÷ channel [7] and acceleration of the hydrolysis of phosphoinositide [10], or activation of protein kinase C [6]. Moreover, it has also been reported that Ang II induces Ca 2÷ efflux from bovine adrenal chromaffin cells through Na+/Ca 2÷ exchange mechanism [8]. Considering these findings, it may be reasonable to speculate that Ang II-induced Ca 2÷ efflux from adult rat cardiomyocytes may be mediated by the mechanism of Na+/Ca 2+ exchange and which may accelerate Ca 2÷ efflux from the cardiomyocytes by the stimulation of inositol triphosphate turnover coupled with ATI receptors. These results are consistent with the report that Ang II stimulates Na+/Ca 2÷ exchanger in rat heart [2]. On the other hand, it has also been reported that inositoi 1,4,5-trisphosphate (IP3), which is produced by the stimulation of inositol triphosphate turnover, increases the [Ca2+]i in cardiomyocytes [3], Ang II may stimulate simultaneously the extrusion of this elevated [Ca2+]i to a resting level as a counter action for increase in [Ca2+]i within the cells. Smith et al. reported that Ang II evoked 4SCa2+ efflux from cultured arterial muscle cells concomitantly with the elevation of [Ca2+]i [17]. However, it is still unclear how Ang II causes the 45Ca 2+ efflux from the cardiomyocytes. We are now investigating the intracellular mechanism which is involved in Ang II-induced Ca 2÷ efflux from freshly isolated adult rat cardiomyocytes. In conclusion, Ang II stimulates Ca 2+ efftux from adult rat cardiomyocytes probably through AT1 receptor. The underlying mechanism, which causes Ca 2+ efflux from the cells is still not entirely understood, however it can be explained at least in part by Na+/Ca 2+ exchange on the plasma membrane. [1] Baker, K.M. and Aceto, J.F., Angiotensin II stimulation of protein synthesis and cell growth in chick heart cells, Am. J. Physiol., 259 (1990) H610-H618.
[2] Ballard, C. and Schaffer, S., Stimulation of the Na+/Ca2+ exchanger by phenylephrine, angiotensin II and endothelin I, J. Mol. Cell. Cardiol., 28 (1996) 11-17. [3] Berridge, M.J., Inositol trisphosphate and calcium signaling, Nature, 361 (1993) 315-325. [4] De Young, M.B. and Scarpa, A., Extracellular ATP induces Ca2+ transients in cardiac myocytes which are potentiated by norepinephrine, FEBS Lett., 223 (1987) 53-58. [5] Downey, LM., Liu, G.S. and Thornton, J.D., Adenosine and the anti-infarct effects of preconditioning, Cardiovasc. Res., 27 (1993) 3-8. [6] Duff, J.L., Marrero, M.B., Paxton, W.G., Schieffer, B., Bemstein, K.E. and Berk, B.C., Angiotensin II signal transduction and the mitogen-activated protein kinase pathway, Cardiovasc. Res., 30 (1995) 511-517. [7] Freer, R.J., Pappano, A.J., Peach, M.J., Bing, K.T., McLean, M.J., Vogel, S. and Sperelakis, N., Mechanism for the positive inotropic effect of angiotensin II on isolated cardiac muscle, Circ. Res., 39 (1976) 178-183. [8] Houchi, H., Okuno, M., Kitamura, K., Ishimura, Y., Ohuchi, T., Tokumura, A. and Oka, M., Stimulatory effect of angiotensin II on calcium efflux from cultured bovine adrenal chromaffin cells, Life Sci., 56 (1995) 109-114. [9] Ishihata, A. and Endoh, M., Species-related differences in inotropic effects of angiotensin II in mammalian ventricular muscle: receptors, subtypes and phosphoinositide hydrolysis, Br. J. Pharmacol., 114 (1995) 447-453. [10] Kem, D.C., Johnson, E.I.M., Capponi, A.M., Chardonnens, D., Lang, U., Blondel, B., Kosida, H. and Vallotton, M.B., Effect of angiotensin 11 on cytosolic free calcium in neonatal rat cardiomyocytes, Am. J. Physiol., 261 (1991) C77-C85. [I 1] Laragh, J.H., The lenin system and new understanding of the complications of hypertension and their treatment, Arzneim.-ForschJ Drug Res., 43 (1993) 247-254. [12] Lewartowski, B., Wolska, B.M. and Zdanowski, K., The effects of blocking the Na-Ca exchange at intervals throughout the physiological contraction-relaxation cycle of single cardiac myocyte, J. Mol. Cell. Cardiol., 24 (1992) 967-976. [13] Maulik, N., Engelman, D.T., Watanabe, M., Engelman, R.M., Maulik, G., Cordis, G.A. and Das, D.K., Nitric oxide signaling in ischemic heart, Cardiovasc. Res., 30 (1995) 593-601. [14] Mitra, R. and Morad, M., A uniform enzymatic method for dissociation of myocytes from hearts and stomachs of vertebrates, Am. J. Physiol., 249 (1985) H1056-H1060. [15] Ogihara, T., Higashimori, K., Masuo, K. and Mikami, H., Pilot study of a new angiotensin II receptor antagonist, TCV-116: effects of a single oral dose on blood pressure in patients with essential hypertension, Ciin. Therapeutics, 15 (1993) 684-691. [16] Sexena, P.R., Interaction between the renin-angioteasin-aldosterone and sympathetic nervous systems, J. Cardiovasc. Pharmacol., 19 (1992) $80-$88. [17] Smith, J.B. and Smith, L., Extracellular Na ÷ dependence of changes in free Ca2÷, 45Ca2+ efflux, and total cell Ca~+ produced by angiotensin 11 in cultured arterial muscle cells, J. Biol. Chem., 262 (1987) 17455-17460. [18] Sneddon, P. and Bumstock, G., ATP as a co-transmitter in rat tail artery, Ear. J. Pharmacol., 106 (1985) 149-152. [19] Timmermans, P.B.M.W.M., Wong, P.C., Chiu, A.T. and Herblin, W.F., Nonpeptide angiotensin 11receptor antagonists, Trends Pharmacol. Sci., 12 (1991) 55-62. [20] Zimmerman, A.N.E. and Hulsmann, W.C., Paradoxical influence of calcium ions on the permeability of the cell membranes of the isolated rat heart, Nature, 211 (1966) 646-647.